Preferential incorporation of glucosamine into the galactosamine moieties of chondroitin sulfates in articular cartilage explants

OBJECTIVE: To determine the metabolic fate of glucosamine (GlcN) in intact articular cartilage tissue. METHODS: Intact articular cartilage explants were cultured for up to 13 days in Dulbecco's modified Eagle's medium supplemented with 1) 1-13C-labeled GlcN, 2) 1-13C-labeled glucose (Glc), or 3) no labeling. Every 3-4 days, samples were removed and frozen in liquid nitrogen for carbon-13 magnetic resonance spectroscopic (MRS) analysis. The metabolic products of the labeled precursors were determined from the MRS data based on resonance positions and comparison with known standards and published values. RESULTS: GlcN was taken up by the chondrocytes and incorporated selectively into the hexosamine, but not the hexuronic acid, components of the glycosaminoglycan chains of articular cartilage proteoglycan. The data also demonstrated that GlcN is the substrate of choice for the galactosamine moieties of the chondroitin sulfates, incorporating at levels 300% higher than with an equivalent amount of labeled Glc. CONCLUSION: The results indicate that GlcN facilitates the production of proteoglycan components that are synthesized through the hexosamine biochemical pathway.     

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.Seasonal influenza outbreaks across the globe cause an estimated 250,000–500,000 deaths annually (1). Current influenza vaccines need to be updated every few years because of antigenic drift (2). Despite intensive monitoring, strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past (2). Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift facilitating a potential pandemic outbreak. These concerns have expedited efforts toward developing a universal influenza vaccine.Neutralizing antibodies (nAbs) against hemagglutinin (HA) are the primary correlate for protection in humans and hence HA is an attractive target for vaccine development (3). The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the disulfide-linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit, whereas the stem domain predominantly comprises the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH-induced conformational rearrangement in the host-cell endosomes to adopt the virus–host membrane fusion-competent state (4, 5).The antigenic sites on the globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed nAbs (6). However, extensive efforts have resulted in the isolation of monoclonal antibodies (mAbs) that bind within the globular head and inhibit receptor attachment, which neutralize drifted variants of an HA subtype or heterosubtypic HA (7–16). The HA stem is targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes (17). The epitopes of these bnAbs in the HA stem are more conserved across different influenza HA subtypes compared with the antigenic sites in the HA globular head (18).During a primary infection, the immunodominant globular head domain suppresses the response toward the conserved stem. Several efforts have been made to circumvent this problem. Repeated immunizations with full-length, chimeric HAs (cHAs) in a protracted vaccination regimen have been shown to boost stem-directed responses in mice (19). Alternatively, full-length HA presented on nanoparticles (np) has been shown to elicit stem-directed nAbs (20). Attempts have also been made to steer the immune response toward the conserved HA stem by hyperglycosylating the head domain (21). Although the aforementioned strategies need to be further evaluated and provide novel alternatives, detrimental interference from the highly variable immunodominant head domain in eliciting a broad functional response cannot be completely evaded. A “headless” stem domain immunogen offers an attractive solution. However, early attempts at expressing the HA2 subunit independently in a native, prefusion conformation were unsuccessful. In the absence of the head domain, the HA2 subunit expressed in Escherichia coli spontaneously adopted the low-pH conformation (22) in which the functional epitopes of stem-directed bnAbs are disrupted. More recently, the entire HA stem region has been expressed in a prefusion, native-like conformation in both prokaryotic and eukaryotic systems adopting multiple strategies (23–26).Design of independently folding HA stem fragments which adopt the prefusion HA conformation presents another approach to elicit bnAbs against influenza (27, 28). The A helix of the HA2 subunit contributes substantial contact surface to the epitope of stem-directed bnAbs such as CR6261, F10, and others. Although multivalent display of A helix on the flock house virus as a virus-like particle platform elicited cross-reactive antibodies, it conferred only minimal protection (20%) against virus challenge in mice (29).We report the design and characterization of engineered headless HA stem immunogens based on the influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation-sensitive, stem-directed bnAbs such as CR6261 (30), F10 (31), and FI6v3 (32) with a high-affinity [equilibrium dissociation constant (K_D) of 10–50 nM]. The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both group 1 (H1 and H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD₉₀) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens confer robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous stem domain immunogens (23–25), the designed immunogens were purified from the soluble fraction in E. coli. The HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free, which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks.     

Validating administratively derived frailty scores for use in Veterans Health Administration emergency departments

Objectives

Frailty is a clinical syndrome characterized by decreased physiologic reserve that diminishes the ability to respond to stressors such as acute illness. Veterans Health Administration (VA) emergency departments (ED) are the primary venue of care for Veterans with acute illness and represent key sites for frailty recognition. As questionnaire-based frailty instruments can be cumbersome to implement in the ED, we examined two administratively derived frailty scores for use among VA ED patients.

Methods

This national retrospective cohort study included all VA ED visits (2017–2020). We evaluated two administratively derived scores: the Care Assessment Needs (CAN) score and the VA Frailty Index (VA-FI). We categorized all ED visits across four frailty groups and examined associations with outcomes of 30-day and 90-day hospitalization and 30-day, 90-day, and 1-year mortality. We used logistic regression to assess the model performance of the CAN score and the VA-FI.

Results

The cohort included 9,213,571 ED visits. With the CAN score, 28.7% of the cohort were classified as severely frail; by VA-FI, 13.2% were severely frail. All outcome rates increased with progressive frailty (p-values for all comparisons < 0.001). For example, for 1-year mortality based on the CAN score frailty was determined as: robust, 1.4%; prefrail, 3.4%; moderately frail, 7.0%; and severely frail, 20.2%. Similarly, for 90-day hospitalization based on VA-FI, frailty was determined as prefrail, 8.3%; mildly frail, 15.3%; moderately frail, 29.5%; and severely frail, 55.4%. The c-statistics for CAN score models were higher than for VA-FI models across all outcomes (e.g., 1-year mortality, 0.721 vs. 0.659).

Conclusions

Frailty was common among VA ED patients. Increased frailty, whether measured by CAN score or VA-FI, was strongly associated with hospitalization and mortality and both can be used in the ED to identify Veterans at high risk for adverse outcomes. Having an effective automatic score in VA EDs to identify frail Veterans may allow for better targeting of scarce resources.     

Tamilnadia uliginosa (Retz) Tirveng and Sastre: Potential Application from Traditional Remedies to Modern Therapeutics

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Tamilnadia uliginosa (Retz) Tirveng and Sastre (Rubiaceae) is a small edible medicinal plant utilized in...     

A Proposed Methodology for In Vitro Evaluation of Bitterness in Drug Solutions and In Vitro Drug Release Samples

Purpose

Ethical and safety concerns, paediatric taste panels and predictivity in early drug development for strategic decisions are some of the reasons for seeking in vitro methods of bitterness evaluation for drugs and drug products. In this study, taste panel studies and in vitro drug release studies have been performed, correlated to each other and proposed as an analytical tool for evaluation of bitterness.

Methods

Bitterness threshold and bitterness scores for different solutions of ondansetron hydrochloride (ONS) were estimated by taste panel studies. In vitro drug release studies on taste-masked drug product in pharmacopoeial apparatus and an in-house-developed apparatus were performed and correlated to drug release studies in oral cavity.

Results

Concentration of 22 μg/ml and below was perceived bitterless by all the volunteers of taste panel. A second-order polynomial equation (y?=?0.6206x ²???0.2011x???0.7796; correlation coefficient R ²?=?0.991) was derived as a relationship between bitterness score and log ONS concentration. Drug release in in-house-assembled apparatus and oral cavity were not statistically different (α?=?0.05) at both 60 and 120 s.

Conclusions

Bitterness threshold and bitterness scores are helpful in evaluation of bitterness in drug solutions and samples obtained from drug release studies.     

Equine trypanosomosis in central and western Punjab: Prevalence,haemato-biochemical response and associated risk factors

The detection of Trypanosoma evansi in blood is intricate, primarily in chronic stage of infection, as the parasitaemia is often low and fluctuating. The climatic conditions of the target area of Punjab (a province of India with a total of 34,000 horses and ponies used for sports and transport) are conducive for the parasite propagation. The objective of present investigation was to assess the prevalence of T. evansi in central and western Punjab by PCR and card agglutination test (CATT/T. evansi) in relation to clinico-haematobiochemical alterations and risk factors associated with latent trypanosomosis. A total of 169 equine blood and serum samples tested by CATT/T. evansi revealed 16 cases positive, with 6.8% from central plain and 13.63% from western zone. To assess the specificity of serological test, PCR₁ was performed using established primer pair TR3 5′-GCG CGG ATT CTT TGC AGA CGA-3′ and TR4 5′-TGC AGA CAC TGG AAT GTT ACT-3′ for T. evansi. PCR₂ applied with primer pair RoTat1.2F: 5′-ATG TCA ACG ATG CCT GTT ACA TTA CGC AC-3′ and RoTat1.2R: 5′-TAA ATA TCA CTG TCA AGA CCT GCT GCG G-3′ to rule out the consensus between the finding of the two PCR assays and agglutination test for T. evansi, which displayed results in concordance with PCR₁. PCR assays showed 1.92 and 1.51% positive samples from central plain and western zone, respectively. With respect to PCR assay, CATT/T. evansi showed 100% sensitivity and 92.1% specificity. Microscopy showed a very low prevalence rate of 0.59% with only one sample positive with teaming parasitaemia. Comparison between sexes revealed higher positivity in mares by the three tests (BSE: 0.95%, PCR: 2.88%, CATT/T. evansi: 14.42%). The haemato-biochemical factors were found to be altered in PCR positive cases, while the mean value of vital parameters lied in normal range in seropositive cases. The female horse (RR = 0.0937, 95% CI = 1.388–190.223%) population was found to be at the highest risk of seropositivity for T. evansi, particularly in the unorganized farms (RR = 19.726, 95% CI = 2.918–400.221%).