RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression

Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract–binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5′ and 3′ ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5′ and the 3′ pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.     

Quantification of Bacterial DNA from Infected Human Root Canals Using qPCR and DAPI after Disinfection with Established and Novel Irrigation Protocols

The removal of bacterial infections within the root canal system is still a challenge. Therefore, the cleansing effect of established and new irrigation-protocols (IP) containing silver diamine fluoride (SDF) 3.8% on the whole root canal system was analyzed using quantitative PCR (qPCR) and 4′,6-diamidino-phenylindole-(DAPI)-staining. Extracted human premolars were instrumented up to F2 (ProTaper Gold) under NaCl 0.9% irrigation and incubated with Enterococcus faecalis for 42 days. Subsequently, different ultrasonically agitated IP were applied to the roots: control (no irrigation), 1. NaOCl 3%, EDTA 20%, CHX 2%, 2. NaOCl 3%, EDTA 20%, 3. NaOCl 3%, EDTA 20%, SDF 3.8%, 4. SDF 3.8%, and 5. NaCl 0.9%. One half of the root was investigated fluorescent-microscopically with DAPI. The other half was grinded in a cryogenic mill and the bacterial DNA was quantified with qPCR. The qPCR results showed a statistically significant reduction of bacteria after the application of IP 1, 2, and 3 compared to the control group. While IP 4 lead to a bacterial reduction which was not significant, IP 5 showed no reduction. These data corresponded with DAPI staining. With qPCR a new molecular-biological method for the investigation of the complete root canal system was implemented. The novel IP 3 had an equally good cleansing effect as the already established IP.     

Mechanical Properties and Root Canal Shaping Ability of a Nickel–Titanium Rotary System for Minimally Invasive Endodontic Treatment: A Comparative In Vitro Study

Selection of an appropriate nickel–titanium (NiTi) rotary system is important for minimally invasive endodontic treatment, which aims to preserve as much root canal dentin as possible. This study aimed to evaluate selected mechanical properties and the root canal shaping ability of TruNatomy (TRN), a NiTi rotary system designed for minimally invasive endodontic shaping, in comparison with existing instruments: HyFlex EDM (HEDM), ProTaper Next (PTN), and WaveOne Gold (WOG). Load values measured with a cantilever bending test were ranked as TRN < HEDM < WOG < PTN (p < 0.05). A dynamic cyclic fatigue test revealed that the number of cycles to fracture was ranked as HEDM > WOG > TRN > PTN (p < 0.05). Torque and vertical force generated during instrumentation of J-shaped artificial resin canals were measured using an automated instrumentation device connected to a torque and vertical force measuring system; TRN exhibited smaller torque and vertical force values in most comparisons with the other instruments. The canal centering ratio for TRN was smaller than or comparable to that for the other instruments except for WOG at the apex level. Under the present experimental conditions, TRN showed higher flexibility and lower torque and vertical force values than the other instruments.     

Structural basis for high-affinity HER2 receptor binding by an engineered protein

The human epidermal growth factor receptor 2 (HER2) is specifically overexpressed in tumors of several cancers, including an aggressive form of breast cancer. It is therefore a target for both cancer diagnostics and therapy. The 58 amino acid residue Zher2 affibody molecule was previously engineered as a high-affinity binder of HER2. Here we determined the structure of Zher2 in solution and the crystal structure of Zher2 in complex with the HER2 extracellular domain. Zher2 binds to a conformational epitope on HER2 that is distant from those recognized by the therapeutic antibodies trastuzumab and pertuzumab. Its small size and lack of interference may provide Zher2 with advantages for diagnostic use or even for delivery of therapeutic agents to HER2-expressing tumors when trastuzumab or pertuzumab are already employed. Biophysical characterization shows that Zher2 is thermodynamically stable in the folded state yet undergoing conformational interconversion on a submillisecond time scale. The data suggest that it is the HER2-binding conformation that is formed transiently prior to binding. Still, binding is very strong with a dissociation constant K_D = 22 pM, and perfect conformational homogeneity is therefore not necessarily required in engineered binding proteins. A comparison of the original Z domain scaffold to free and bound Zher2 structures reveals how high-affinity binding has evolved during selection and affinity maturation and suggests how a compromise between binding surface optimization and stability and dynamics of the unbound state has been reached.     

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ₁34.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ₁34.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ₁34.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ₁34.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.

Herpes simplex viruses (HSV) are human pathogens that switch between lytic and latent infections intermittently (1, 2). This is a lifelong source of infectious viruses (1, 2), in which immediate early proteins drive the onset of HSV replication. Among them, ICP0 enables viral gene expression or reactivation from latency (2–4), which involves chromatin remodeling of the HSV genome, resulting in de novo virus production. In this process, the accessory factor γ₁34.5 of HSV is thought to govern viral protein synthesis (5, 6). It has long been known that γ₁34.5 precludes translation arrest mediated by double-stranded RNA–dependent protein kinase PKR (7–9). The γ₁34.5 protein has also been shown to dampen intracellular nucleic acid sensing, inhibit autophagy, and facilitate virus nuclear egress (10–17). In experimental animal models, wild-type HSV, but not HSV that lacks the γ₁34.5 gene, replicates competently, penetrates from the peripheral tissues to the nervous system and reactivates from latency (18–23). Despite these observations, active HSV replication or reactivation from latency is not readily reconciled by the currently known functions of the γ₁34.5 protein (8–13, 16, 17).Several lines of work demonstrate that tripartite motif (TRIM) proteins regulate innate immune signaling and cell intrinsic resistance to virus infections (24, 25). These host factors typically work as E3 ubiquitin ligases that can synthesize degradative or nondegradative ubiquitination on viral or host proteins. A number of TRIM proteins, for example TRIM5α, TRIM19, TRIM21, TRIM22, and TRIM43, act at different steps of virus replication and subsequently inhibit viral production (26–32). Recent evidence indicates that TRIM23 limits the replication of certain RNA viruses and DNA viruses, including HSV-1 (33). In doing so, TRIM23 recruits TANK-binding kinase 1 (TBK1) to autophagosomes, thus promoting TBK1-mediated phosphorylation and activation of the autophagy receptor p62 and ultimately leading to autophagy. It is unknown whether TRIM23 plays an additional role(s) in HSV infection.Here, we report that ICP0 expression is regulated by the γ₁34.5 protein and TRIM23 during HSV-1 infection. We show that TRIM23 facilitates the proteasomal degradation of ICP0, whereas viral γ₁34.5 maintains steady-state ICP0 expression by preventing K27-linked TRIM23 autoubiquitination that is required for TRIM23 activation. The γ₁34.5 protein also interacts with and stabilizes ICP0, enabling productive infection. Furthermore, we provide evidence that TRIM23 binds to ICP0 and induces its K11-linked polyubiquitination, which triggers K48-linked polyubiquitin-dependent proteasomal degradation of ICP0. These insights establish a model of posttranslational networks in which virus- and host-mediated mechanisms regulate immediate early protein ICP0 stability and thereby lytic HSV replication.     

Positive regulation by small RNAs and the role of Hfq

Bacterial small noncoding RNAs carry out both positive and negative regulation of gene expression by pairing with mRNAs; in Escherichia coli, this regulation often requires the RNA chaperone Hfq. Three small regulatory RNAs (sRNAs), DsrA, RprA, and ArcZ, positively regulate translation of the sigma factor RpoS, each pairing with the 5′ leader to open up an inhibitory hairpin. In vitro, rpoS interaction with sRNAs depends upon an (AAN)₄ Hfq-binding site upstream of the pairing region. Here we show that both Hfq and this Hfq binding site are required for RprA or ArcZ to act in vivo and to form a stable complex with rpoS mRNA in vitro; both were partially dispensable for DsrA at 37 °C. ArcZ sRNA is processed from 121 nt to a stable 56 nt species that contains the pairing region; only the 56 nt ArcZ makes a strong Hfq-dependent complex with rpoS. For each of these sRNAs, the stability of the sRNA•mRNA complexes, rather than their rate of formation, best predicted in vivo activity. These studies demonstrate that binding of Hfq to the rpoS mRNA is critical for sRNA regulation under normal conditions, but if the stability of the sRNA•mRNA complex is sufficiently high, the requirement for Hfq can be bypassed.     

Unraveling the Phase Stability and Physical Property of Modulated Martensite in Ni2Mn1.5In0.5 Alloys by First-Principles Calculations

Large magnetic field-induced strains can be achieved in modulated martensite for Ni-Mn-In alloys; however, the metastability of the modulated martensite imposes serious constraints on the ability of these alloys to serve as promising sensor and actuator materials. The phase stability, magnetic properties, and electronic structure of the modulated martensite in the Ni₂Mn_1.5In_0.5 alloy are systematically investigated. Results show that the 6M and 5M martensites are metastable and will eventually transform to the NM martensite with the lowest total energy in the Ni₂Mn_1.5In_0.5 alloy. The physical properties of the incommensurate 7M modulated martensite (7M–IC) and nanotwinned 7M martensite (7M−(52¯)2) are also calculated. The austenite (A) and 7M−(52¯)2 phases are ferromagnetic (FM), whereas the 5M, 6M, and NM martensites are ferrimagnetic (FIM), and the FM coexists with the FIM state in the 7M–IC martensite. The calculated electronic structure demonstrates that the splitting of Jahn–Teller effect and the strong Ni–Mn bonding interaction lead to the enhancement of structural stability.     

Characterization of SARS-CoV-2 Evasion: Interferon Pathway and Therapeutic Options

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. SARS-CoV-2 is characterized by an important capacity to circumvent the innate immune response. The early interferon (IFN) response is necessary to establish a robust antiviral state. However, this response is weak and delayed in COVID-19 patients, along with massive pro-inflammatory cytokine production. This dysregulated innate immune response contributes to pathogenicity and in some individuals leads to a critical state. Characterizing the interplay between viral factors and host innate immunity is crucial to better understand how to manage the disease. Moreover, the constant emergence of new SARS-CoV-2 variants challenges the efficacy of existing vaccines. Thus, to control this virus and readjust the antiviral therapy currently used to treat COVID-19, studies should constantly be re-evaluated to further decipher the mechanisms leading to SARS-CoV-2 pathogenesis. Regarding the role of the IFN response in SARS-CoV-2 infection, in this review we summarize the mechanisms by which SARS-CoV-2 evades innate immune recognition. More specifically, we explain how this virus inhibits IFN signaling pathways (IFN-I/IFN-III) and controls interferon-stimulated gene (ISG) expression. We also discuss the development and use of IFNs and potential drugs controlling the innate immune response to SARS-CoV-2, helping to clear the infection.     

MORC2 gene de novo mutation leads to Charcot–Marie–Tooth disease type 2Z: A pediatric case report and literature review

Rationale:Mutations of the MORC2 gene have most commonly been associated with autosomal-dominant Charcot–Marie–Tooth disease type 2Z (CMT 2Z), while the impact of MORC2 mutations in CMT 2Z on neuronal biology and their phenotypic consequences in patients remain to be clarified.Patient concerns:We reported a 27-month-old child with a developmental lag of more than 1 year. He had progressive fatigue for 4 months, accompanied by dysphagia, choking while eating, and progressive aggravation. A genetic study revealed a de novo variant of MORC2, which has not yet been reported.Diagnosis:According to the child''s clinical manifestations, genetic pattern, and American College of Medical Genetics and Genomics pathogenicity analysis, the patient was diagnosed with CMT 2Z caused by MORC2 gene mutation.Interventions:Mitochondrial cocktail therapy (arginine, vitamin B1 tablets, vitamin B2 tablets, coenzyme Q10 capsules, L-carnitine oral liquid, idebenone tablets, etc) was given.Outcomes:Mitochondrial cocktail therapy did not significantly improve the child''s condition, head magnetic resonance imaging lesions were not significantly improved at outpatient follow-up more than 1 month later, and the lesions were basically unchanged.Lessons:The clinical manifestations of the disease were similar to those of Leigh syndrome, and they were not significantly improved by cocktail therapy. This site has not been reported in the literature domestically or abroad, and the pathogenesis of CMT 2Z caused by this site mutation is indeed not related to mitochondrial dysfunction. Our study is helpful for clinicians with regard to the differential diagnosis of Leigh syndrome and CMT 2Z and improvement of clinicians’ understanding of CMT 2Z disease.     

A therapeutic convection–enhanced macroencapsulation device for enhancing β cell viability and insulin secretion

Islet transplantation for type 1 diabetes treatment has been limited by the need for lifelong immunosuppression regimens. This challenge has prompted the development of macroencapsulation devices (MEDs) to immunoprotect the transplanted islets. While promising, conventional MEDs are faced with insufficient transport of oxygen, glucose, and insulin because of the reliance on passive diffusion. Hence, these devices are constrained to two-dimensional, wafer-like geometries with limited loading capacity to maintain cells within a distance of passive diffusion. We hypothesized that convective nutrient transport could extend the loading capacity while also promoting cell viability, rapid glucose equilibration, and the physiological levels of insulin secretion. Here, we showed that convective transport improves nutrient delivery throughout the device and affords a three-dimensional capsule geometry that encapsulates 9.7-fold-more cells than conventional MEDs. Transplantation of a convection-enhanced MED (ceMED) containing insulin-secreting β cells into immunocompetent, hyperglycemic rats demonstrated a rapid, vascular-independent, and glucose-stimulated insulin response, resulting in early amelioration of hyperglycemia, improved glucose tolerance, and reduced fibrosis. Finally, to address potential translational barriers, we outlined future steps necessary to optimize the ceMED design for long-term efficacy and clinical utility.

Diabetes mellitus currently burdens over 387 million people worldwide, of which 5 to ∼10% are accounted by patients with type 1 diabetes (T1D) (1). T1D is characterized by the immune destruction of insulin-secreting β cells and the loss of glycemic regulation. Although intensive insulin injection regimens and the use of glucose monitors have been shown to effectively regulate blood glucose, patients are still unable to meet glycemic control targets. In particular, those with severe hypoglycemic events and glycemic lability cannot be effectively stabilized with these technologies (2). In 2000, the Edmonton protocol was developed as a procedure that directly infuses pancreatic islets, isolated from cadaveric donors, into the portal vein of T1D patients. This procedure led to insulin independence in patients for a short period postinfusion (3, 4). However, poor long-term graft survival due to alloimmune and autoimmune rejections and engraftment inefficiency prevents sustained, therapeutic effects (5–7). Although immunosuppressants are coadministered with the transplanted cells to prevent graft rejection, 56% of patients experience partial to complete graft loss after 1 y, and only 10% of patients remain insulin independent after 5 y (4, 8). The majority of patients also experience complications from immunosuppression, including elevated risk of opportunistic infections and cancer (4, 8, 9). In addition, islet transplantation is burdened by a major islet donor shortage, since often two or more human pancreases are needed to achieve a sufficient number of islets (10).The complications of immune rejection could be overcome with macroencapsulation devices (MEDs), in which glucose-sensing, insulin-secreting cell sources like pluripotent, stem cell–derived β clusters (SC-βCs) (11), or other islet sources, are transplanted within an immune-isolating vehicle to promote cell survival and function. In MEDs, islets are housed in a single compartment that selectively permits the exchange of nutrients while obstructing host immune effectors such as cells and antibodies. Over the past few decades, MEDs have successfully restored insulin independence and normoglycemia in T1D animal models (10). However, scaling these devices for human applications has been challenging. Currently, passive diffusion-based MEDs, including Encaptra, βAir Βio-Artificial Pancreas, Cell Pouch, and MAILPAN, are being explored in phase I/II clinical trials (12–14). Nevertheless, these diffusion-based devices still suffer from limitations in the transport of glucose, insulin, and other biomolecules to the core of these devices, which compromise the survival and function of encapsulated cells. Ultimately, these devices are restricted in geometry, thickness, and cell-loading capacity.More specifically, a significant portion of encapsulated cells become nonviable immediately after transplantation because of the lack of vascularization, which results in hypoxia and limited nutrient availability. Thus, during the initial prevascularization period, which lasts ∼14-d posttransplantation, solute exchange and insulin secretion cannot occur effectively using conventional MEDs (15–17). For this reason, many encapsulated cells prematurely lose their function and eventually die. Various strategies have been developed to expediate angiogenesis around the device, especially during the initial hypoxic period after device transplantation, to reduce cell loss. Examples include prevascularization of the device, infusion of vascular endothelial growth factor, and cotransplantation of mesenchymal stem cells (15, 18–21). In another instance, βAir Βio-Artificial Pancreas incorporated a daily refillable oxygen chamber in between two islet slabs to maintain adequate oxygen supply, but the chamber is 15- to 30-fold thicker than islet layers. Despite improvements in cell viability, these strategies still cannot guarantee adequate glucose sensing and insulin release kinetics of the islets, and they further limit the available space for cell packing (22).To supply cells with enough nutrients, it has been suggested that the islet density of the MEDs should be set to 5 to ∼10% of the volume fraction (23). Consequently, a limited mass of islets must be placed within a large device to ensure optimal nutrient distribution. Otherwise, devices exhibit extreme cell loss. For instance, TheraCyte, which packs 70 to ∼216 islet equivalent (IEQ) in 4.5 μL or 1,000 IEQ in 40 μL volume, exhibited poor cell survival (19). The remaining cells were neither capable of restoring euglycemia in rodents (1,000 to ∼2,000 IEQ required) nor sustaining a therapeutic dosage needed for humans (∼500,000 islets) in a reasonably sized device (13, 24–26).To overcome these nutrient delivery challenges and improve cell-loading capacity, we designed a convection-enhanced MED (ceMED) to perfuse the device continuously. We hypothesized that convective nutrient transport in ceMED would 1) deliver more nutrients compared with diffusion-based devices, 2) increase cell survival beyond the distance limit of diffusion, 3) support a three-dimensional (3D), expanded cell layer to increase the loading capacity, 4) improve glucose sensitivity and timely insulin secretion via faster biomolecule transport, and 5) show efficacy in vivo by reducing hyperglycemia before vascularization. Overall, we demonstrated that the convective motion promotes survival of insulin-secreting β cells encapsulated at high density. We also demonstrated that it effectively captures the dynamics of glucose concentrations in the transplantation site, resulting in more appropriate insulin secretion with faster on/off responses. Finally, the ceMED showed early, vascular-independent reduction in blood glucose levels in hyperglycemic rat models several days prior to the critical 14-d posttransplantation.     

White patients’ physical responses to healthcare treatments are influenced by provider race and gender

The healthcare workforce in the United States is becoming increasingly diverse, gradually shifting society away from the historical overrepresentation of White men among physicians. However, given the long-standing underrepresentation of people of color and women in the medical field, patients may still associate the concept of doctors with White men and may be physiologically less responsive to treatment administered by providers from other backgrounds. To investigate this, we varied the race and gender of the provider from which White patients received identical treatment for allergic reactions and measured patients’ improvement in response to this treatment, thus isolating how a provider’s demographic characteristics shape physical responses to healthcare. A total of 187 White patients experiencing a laboratory-induced allergic reaction interacted with a healthcare provider who applied a treatment cream and told them it would relieve their allergic reaction. Unbeknownst to the patients, the cream was inert (an unscented lotion) and interactions were completely standardized except for the provider’s race and gender. Patients were randomly assigned to interact with a provider who was a man or a woman and Asian, Black, or White. A fully blinded research assistant measured the change in the size of patients’ allergic reaction after cream administration. Results indicated that White patients showed a weaker response to the standardized treatment over time when it was administered by women or Black providers. We explore several potential explanations for these varied physiological treatment responses and discuss the implications of problematic race and gender dynamics that can endure “under the skin,” even for those who aim to be bias free.

The face of medicine is changing. Women and people of color make up an increasing percentage of health care providers (1–3). In 2017, for the first time in history, women were the majority of accepted medical school applicants in the United States and the number of non-White accepted applicants rose to above 50%. Here, we ask whether this recent demographic shift in the race and gender of doctors is also shifting long-held, societally pervasive notions of what a doctor “looks like.”Despite the increasing diversity of the medical field, for most people in most contexts, the association between “doctor” and “White man” is still likely strong and pervasive. This is hardly surprising. For most of medical history in North America, the majority of physicians fit this profile (see Fig. 1 A and B), and even now the majority of practicing physicians are still men and nearly half are White (see Fig. 1 C and D). Consequently, the emerging links between “Doctor and Woman” and between “Doctor and Black person,” for example, are likely weak. Moreover, to the extent that those associations exist, they are likely to have to compete for attention with an array of strong, frequent, and negative associations that undermine the links between women and competence and African Americans and competence (4–6).Open in a separate windowFig. 1.The change in the representation of women (A) and people color (B) in the number of accepted applicants to US medical schools, as well as the current representation of professionally active women physicians (C) and physicians of color (D). (A and B) From the Association of American Medical Colleges (AAMC). (C) From the Henry J. Kaiser Family Foundation. (D) From 2013 from the Association of American Medical Colleges (AAMC). AAMC data on race/ethnicity were not available for 2013 or 2014, hence explaining the gaps in the graph around these years in B.In patient–provider interactions, as in every social encounter, people bring with them a set of learned associations about social groups that have been formed by their various life experiences (e.g., personal interactions, media exposure) (6–12). Mirroring the historical representation of doctors in actual medical practice, representations of doctors in popular media have overwhelmingly been as White men (13–15). Patients who have learned this societally pervasive “Doctor = White man” association through their actual encounters with physicians as well as through movies, television, books, and advertising may respond less positively to care from Black and women providers. These associations may exist at an implicit level even in the context of positive explicit attitudes toward Black doctors and women doctors (16, 17), and they are potentially powerful, influencing the course of medical care. Also, while it is clear from past research that being a target of bias can be harmful to health (e.g., people who face race-based discrimination face adverse physical and mental health consequences) (18), it is unclear whether viewing another social group in light of societally pervasive associations (e.g., about doctors on the basis of gender and race) can be harmful to the health of the perceiver.Here, we focus on how the race and gender of doctors may impact patients’ responses to the expectations doctors set about medical treatment. Previous research shows that a provider’s expressed expectations for a medical treatment (i.e., that it will benefit patients) can improve patient engagement, adherence, and physiological responses to treatment (19–25). Based on these findings, we anticipate that patients who interact with a doctor whose personal characteristics (e.g., race, gender) do not conform to dominant societal representations of what a doctor looks like may be less responsive to such expectations. We hypothesize that patients may be less responsive to the exact same medical treatment when the doctor who sets expectations that this treatment will be beneficial is not a White man.This hypothesis draws on a large and growing body of research suggesting that the total effect of a healthcare treatment depends on the social context in which that treatment takes place (25–29). The realization that the social context can influence treatment and medical outcomes is bolstered by a large body of research on the placebo effect (26). Although people may sometimes assume that actual pharmaceutical properties of a medication or treatment are solely responsible for its total benefit, placebo paradigms show that the total effect of treatment is in fact a combined product of the drug and their medical properties (e.g., acetaminophen, antihistamines), the body’s natural healing abilities (e.g., endogenous opioids and antihistamines), and the psychological and social context (e.g., what a patient believes about treatment and the qualities of the person who administers the treatment) (SI Appendix, Fig. S1). For example, past research suggests that a physician’s characteristics, such as their projected warmth and competence, influences how much a patient improves in response to treatment. In one recent study (22), the researchers independently manipulated whether a provider acted more or less warm, and more or less competent, toward a patient during an allergy skin prick test that induced a mild allergic reaction. The provider set positive expectations about a placebo cream (i.e., unscented hand lotion) placed on the reaction, informing patients that this cream was an antihistamine that would reduce the reaction. When the provider was both warm and competent, patients showed a stronger physiological response to the placebo treatment over time; their allergic reaction decreased the most rapidly in size, in response to the positive expectations that the provider had set. Thus, aspects of social interactions with providers can influence the degree to which the positive expectations that a provider sets about treatment ultimately influence physiological treatment response.As in most social interactions in the United States, race and gender are likely salient aspects of the social context in patient–provider interactions (30, 31, 32). Previous research has found, for example, that patient race can influence the quality of care received from doctors in myriad ways (33–36). Here, we focus on provider race and provider gender as features of the social context that can influence patients’ response to treatment. Specifically, we ask the following: will White patients exhibit a weaker physiological response to the expectations set about treatment by doctors who are not White and men?     

Structural and energetic analysis of metastable intermediate states in the E1P–E2P transition of Ca2+-ATPase

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase transports two Ca²⁺ ions from the cytoplasm to the SR lumen against a large concentration gradient. X-ray crystallography has revealed the atomic structures of the protein before and after the dissociation of Ca²⁺, while biochemical studies have suggested the existence of intermediate states in the transition between E1P⋅ADP⋅2Ca²⁺ and E2P. Here, we explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. The simulations suggest that a series of structural changes accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca²⁺ ions toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization. Free energy profiles of the transition assuming different protonation states suggest rapid exchanges between Ca²⁺ ions and protons when the Ca²⁺ ions are released toward the SR lumen.

Sarcoplasmic reticulum Ca²⁺-ATPase (SR Ca²⁺-ATPase or SERCA1a) is a representative P-type ATPase that transports Ca²⁺ ions against a 10⁴ times concentration gradient across the SR membrane. The transport mechanism was originally described by E1/E2 theory, whereby the protein alternates between Ca²⁺ high-affinity E1 and low-affinity E2 states. A more-detailed reaction cycle requires multiple steps, including the binding/dissociation of Ca²⁺, H⁺-counter transport, ATP-binding and hydrolysis, phosphorylation/dephosphorylation of Asp351, and the dissociation of ADP and Pi (1–3) (SI Appendix, Fig. S1). Structurally, the Ca²⁺-ATPase consists of three cytoplasmic domains (actuator; A, nucleotide-binding; N, and phosphorylation; P) and 10 transmembrane (TM) helices (M1-M10) (4, 5). Two Ca²⁺-binding sites are located in M4-M6 and M8 (6), while a nucleotide, ATP or ADP, is bound at the N–P domain interface (7). Functional interconnections between the cytoplasmic domains and TM helices are necessary in the reaction cycle (8, 9).Molecular mechanisms underlying Ca²⁺ uptake by the ATPase have been investigated in biochemical experiments as well as structural studies. In particular, crystal structures of the Ca²⁺-ATPase, which represent different physiological states in the cycle (SI Appendix, Fig. S1), have provided essential information for understanding structure–function relationships (8, 9). Each crystal structure well explains the results of mutagenesis (3, 10), limited proteolysis studies (11, 12), and other biochemical experiments. Comparisons between multiple crystal structures provide direct evidence on how conformational changes of the Ca²⁺-ATPase take place from one step to another in the cycle. For instance, crystal structures that represent E1P⋅ADP⋅2Ca²⁺ and E2P (Fig. 1 A and B) reveal important conformational changes to release Ca²⁺ toward the SR lumen: 1) the A-domain rotates ∼90°; 2) the threonine–glycine–glutamate–serine (TGES) loop in the A-domain reaches the phosphorylated Asp351 in the P-domain (13–15); 3) M1-M6 are rearranged to open the luminal gate for the dissociation of Ca²⁺. Despite the increasing structural information, there are still unresolved questions. A series of biochemical studies suggested the existence of two intermediate states, E1P⋅2Ca²⁺ and E2P⋅2Ca²⁺. However, atomistic structures and their energetics in these intermediate states would be required to understand their functional roles.Open in a separate windowFig. 1.Structures of SR Ca²⁺-ATPase embedded in a DOPC membrane in E1P⋅ADP⋅2Ca²⁺ (PDB ID: 2ZBD) (A) and E2P (PDB ID: 2ZBE) (B). The three cytoplasmic domains (A, N, and P) are colored in red, purple, and green, respectively. (C) A structural transition pathway predicted using the mean-force string method is projected onto a two-dimensional map along with the two distance RMSDs (dRMS) from the representative structures in MD simulations of E1P and E2P_dp. In the simulation, Glu908 at the Ca²⁺-binding sites is protonated to mimic the same protonation states of E1P. Only the atomic coordinates that are involved in the collective variables (CVs) are used for dRMS calculations. The five substates (SSs) are defined along the pathway via the fixed radius clustering method (1 to 17: red; 18 to 27: yellow; 28 to 35: green; 36 to 51: cyan; and 52 to 64: blue). Among the 64 images, 5 images (10, 23, 31, 44, and 57) are selected as five representative SSs.There are several computational tools to predict conformational transition pathways of proteins, such as morphing (16), normal mode analysis (17), or molecular dynamics (MD) simulation based on coarse-grained or atomistic models (18–21). However, large conformational changes of the Ca²⁺-ATPase happen on the milliseconds or slower time scales, which are not accessible in brute-force MD simulations (18–21) even when using MD-specialized supercomputers, such as Anton/Anton 2 (22, 23) or MDGRAPE-4A (24). In this study, we perform atomistic MD simulations with an enhanced conformational sampling method to investigate the conformational pathway and free energy profile in the transition between E1P⋅ADP⋅2Ca²⁺ and E2P. We utilize the mean-force string method (25) for obtaining one of the most probable transition pathways. The free energy profile along the pathway is then calculated with umbrella sampling (26). The same approach has been previously applied to adenylate kinase in solution (27) and multidrug transporter AcrB (28) and the ABC heme transporter (29) in biological membranes. A similar method, the string method with swarm trajectories (30), has been applied to several membrane proteins (31, 32). Das et al. applied the method to four conformational transitions of the Ca²⁺-ATPase, including the same step examined in the current study (33).In the current simulation study, we intend to compare the simulation results with those of existing structural and biochemical studies. In particular, the two intermediate states (E1P⋅2Ca²⁺ and E2P⋅2Ca²⁺) and the Ca²⁺-ATPase–lipid interactions in the E1P–E2P transition are examined using the simulation trajectories. The predicted interactions between phospholipids and basic sidechains of Ca²⁺-ATPase are compared with recent X-ray crystallography studies (34). We also investigate the effect of protonation states in the E1P–E2P transitions from atomistic MD simulations, which is difficult via experimental studies. By integrating structural, biochemical, and computational results, we shed light on the structural and energetic features of the E1P–E2P transition of the Ca²⁺-ATPase in unprecedented detail.     

Amorphous–Crystalline Calcium Phosphate Coating Promotes In Vitro Growth of Tumor-Derived Jurkat T Cells Activated by Anti-CD2/CD3/CD28 Antibodies

A modern trend in traumatology, orthopedics, and implantology is the development of materials and coatings with an amorphous–crystalline structure that exhibits excellent biocopatibility. The structure and physico–chemical and biological properties of calcium phosphate (CaP) coatings deposited on Ti plates using the micro-arc oxidation (MAO) method under different voltages (200, 250, and 300 V) were studied. Amorphous, nanocrystalline, and microcrystalline statesof CaHPO₄ and β-Ca₂P₂O₇ were observed in the coatings using TEM and XRD. The increase in MAO voltage resulted in augmentation of the surface roughness R_a from 2.5 to 6.5 µm, mass from 10 to 25 mg, thickness from 50 to 105 µm, and Ca/P ratio from 0.3 to 0.6. The electrical potential (EP) of the CaP coatings changed from −456 to −535 mV, while the zeta potential (ZP) decreased from −53 to −40 mV following an increase in the values of the MAO voltage. Numerous correlations of physical and chemical indices of CaP coatings were estimated. A decrease in the ZP magnitudes of CaP coatings deposited at 200–250 V was strongly associated with elevated hTERT expression in tumor-derived Jurkat T cells preliminarily activated with anti-CD2/CD3/CD28 antibodies and then contacted in vitro with CaP-coated samples for 14 days. In turn, in vitro survival of CD4⁺ subsets was enhanced, with proinflammatory cytokine secretion of activated Jurkat T cells. Thus, the applied MAO voltage allowed the regulation of the physicochemical properties of amorphous–crystalline CaP-coatings on Ti substrates to a certain extent. This method may be used as a technological mechanism to trigger the behavior of cells through contact with micro-arc CaP coatings. The possible role of negative ZP and Ca²⁺ as effectors of the biological effects of amorphous–crystalline CaP coatings is discussed. Micro-arc CaP coatings should be carefully tested to determine their suitability for use in patients with chronic lymphoid malignancies.