Evidence that drug flux across synthetic membranes is described by normally distributed permeability coefficients.

Over recent decades, the use of in vitro diffusion cell studies to assess skin permeability has evolved into a major research tool, providing key insights into the relationships between skin, drug and formulation. Sometimes, such studies involve synthetic membranes as this approach can yield useful inferences with respect to drug-skin partitioning and diffusion phenomena. Yet despite the popularity of such studies, it is still not at all known whether typical solute transport across synthetic barriers results in a normal distribution of permeability coefficients or alternatively some type of skewed distribution. The present study aims to shed light on this issue. To this end, five compounds (testosterone, oestradiol, corticosterone, aldosterone and adenosine) exhibiting a broad range of octanol-water partition coefficient values were selected as test penetrants. The protocol involved taking multiple replicate measurements of each drug's passive steady state flux through poly(dimethylsiloxane) membrane. Each penetrant's resultant permeability coefficient database was subjected to a Kolmogorov-Smirnov (KS) test for normality. It was found that the permeability coefficients of all five drugs were distributed in a Gaussian-normal fashion. The theoretical significance and practical impact of these findings are discussed.     

IL-1 is required for tumor invasiveness and angiogenesis

Here, we describe that microenvironmental IL-1 beta and, to a lesser extent, IL-1 alpha are required for in vivo angiogenesis and invasiveness of different tumor cells. In IL-1 beta knockout (KO) mice, local tumor or lung metastases of B16 melanoma cells were not observed compared with WT mice. Angiogenesis was assessed by the recruitment of blood vessel networks into Matrigel plugs containing B16 melanoma cells; vascularization of the plugs was present in WT mice, but was absent in IL-1 beta KO mice. The addition of exogenous IL-1 into B16-containing Matrigel plugs in IL-1 beta KO mice partially restored the angiogenic response. Moreover, the incorporation of IL-1 receptor antagonist to B16-containing plugs in WT mice inhibited the ingrowth of blood vessel networks into Matrigel plugs. In IL-1 alpha KO mice, local tumor development and induction of an angiogenic response in Matrigel plugs was less pronounced than in WT mice, but significantly higher than in IL-1 beta KO mice. These effects of host-derived IL-1 alpha and IL-1 beta were not restricted to the melanoma model, but were also observed in DA/3 mammary and prostate cancer cell models. In addition to the in vivo findings, IL-1 contributed to the production of vascular endothelial cell growth factor and tumor necrosis factor in cocultures of peritoneal macrophages and tumor cells. Host-derived IL-1 seems to control tumor angiogenesis and invasiveness. Furthermore, the anti-angiogenic effects of IL-1 receptor antagonist, shown here, suggest a possible therapeutic role in cancer, in addition to its current use in rheumatoid arthritis.     

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein’s folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein’s functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.All proteins are biosynthesized as linear chains, and most need to fold into 3D structures to function. Studies on protein folding in buffers have revealed that a kinetic competition typically exists between protein folding, misfolding, and aggregation. It is the role of the protein homeostasis or proteostasis network in each subcellular compartment to regulate this competition and keep the folded and functional proteome within the physiological concentration range, while minimizing misfolding and aggregation in the face of stresses (1–4). It remains a challenge to discern how the proteostasis network affects the folding of proteins into biologically active conformations required for function in vivo (5).Current methodologies allow for quantification of the partitioning of a protein of interest (POI) between soluble and aggregated states but cannot determine the proportion of the soluble population that is properly folded and functional. Published folding probes have the potential to report on the folded fraction in cells or cell lysate (6–9); however, the extent to which they shift folding equilibria and quantify the folded and functional fraction faithfully has not been studied. Herein, we create POI folding probes by adapting the principle of activity-based protein profiling (10) to quantify the soluble folded and functional fraction of a particular protein in a cell lysate. We seek folding probes that bind to and selectively react with only the folded and functional state of a POI in a cell, leaving the nonfunctional states and other cellular proteins unmodified (Fig. 1A).Open in a separate windowFig. 1.A small molecule folding probe strategy to quantify the soluble folded and functional fraction of a POI in a cell lysate. (A) Overview of the general strategy to selectively covalently label a folded and functional POI without labeling its nonfunctional conformations and other cellular proteins. (B) The experimental scheme to quantify the ratio of the soluble POI that is functional (R_f).Fluorescent folding probes for the de novo-designed enzyme, retroaldolase (RA) (11), and fluorogenic folding probes (12) for the nonenzyme protein, transthyretin (TTR), were developed and scrutinized. We show that destabilized mutant RA and TTR proteins partition into folded and functional as well as misfolded soluble conformations and that this partitioning is sensitive to proteostasis network perturbations. Experiments show that a snapshot of the distribution between folded and functional vs. soluble and misfolded conformational states can be preserved during the small molecule folding probe labeling period, provided that the cellular chaperone holdase activity is sufficient, achieved by rapid ATP depletion in parallel with cell lysis. Sufficient chaperone/chaperonin holdase activity minimizes changes in the folded and functional concentration associated with probe binding and reaction with the POI and renders the relative folding and conjugation rates much less influential.     

Complications,outcomes, and need for fusion after minimally invasive posterior cervical foraminotomy and microdiscectomy

Background context

Posterior cervical foraminotomy (PCF) with or without microdiscectomy (posterior cervical discectomy [PCD]) is a frequently used surgical technique for cervical radiculopathy secondary to foraminal stenosis or a laterally located herniated disc. Currently, these procedures are being performed with increasing frequency using advanced minimally invasive techniques. Although the safety and efficacy of minimally invasive PCF/PCD (MI-PCF/PCD) have been established, reports on long-term outcome and need for secondary surgical intervention at the index or adjacent level are lacking.

Purpose

To determine the rates of complications, long-term outcomes, and need for secondary surgical intervention at the index or adjacent level after MI-PCF and microdiscectomy.

Study design

Retrospective analysis of a prospective cohort.

Patient sample

Seventy patients treated with MI-PCF and/or MI-PCD for cervical radiculopathy.

Outcome measures

Visual Analog Scale for neck/arm (VAS_N/A) pain and Neck Disability Index (NDI).

Methods

Ninety-seven patients underwent MI-PCF with or without MI-PCD between 2002 and 2011. Adequate prospective follow-up was available for 70 patients (95 cervical levels). The primary outcome assessed was need for secondary surgical intervention at the index or adjacent level. The secondary outcomes assessed included complications and improvements in NDI and VAS_N/A scores. All complications were reviewed. Mixed-model analyses of variance with random subject effects and autoregressive first-order correlation structures were used to test for differences among NDI, VAS_A, and VAS_N measurements made over time while accounting for the correlation among repeated observations within a patient. All statistical hypothesis tests were conducted at the 5% level of significance.

Results

Patients were followed for a mean of 32.1 months. Of 70 patients operated, there were 3 (4.3%) complications (1 cerebrospinal fluid leak, 1 postoperative wound hematoma, and 1 radiculitis), none of which required a secondary operative intervention. Five patients required an anterior cervical discectomy and fusion (eight total levels fused) on average 44.4 months after the index surgery. Of those, five (5.3%) were at the index level and three (2.1%) were at adjacent levels. Neck Disability Index scores improved significantly (p<.0001) immediately postoperatively and continued to decrease gradually with time. Visual Analog Scale for neck/arm scores improved significantly (p<.0001) from baseline immediately postoperatively but tended to plateau with time.

Conclusions

Minimally invasive PCF with or without MI-PCD is an excellent alternative for cervical radiculopathy secondary to foraminal stenosis or a laterally located herniated disc. There is a low rate (1.1% per index level per year) of future index site fusion and a very low rate (0.9% per adjacent level per year) of adjacent-level disease requiring surgery.     

HBV-RNA,Quantitative HBsAg,Levels of HBV in Peripheral Lymphocytes and HBV Mutation Profiles in Chronic Hepatitis B

A comprehensive characterization of chronic HBV (CHB) patients is required to guide therapeutic decisions. The cumulative impact of classical and novel biomarkers on the clinical categorization of these patients has not been rigorously assessed. We determined plasma HBV-RNA and HBsAg levels, HBV in peripheral lymphocytes (PBMCs) and HBV mutation profiles in CHB patients. Patient demographics (n = 139) and classical HBV biomarkers were determined during a clinical routine. HBV-RNA in plasma and HBV-DNA in PBMCs were determined by RT-PCR. HBsAg levels were determined using Architect. In samples with HBV-DNA viral load >1000 IU/mL, genotype mutations in precore (PC), basal core promoter (BCP), HBsAg and Pol regions were determined by sequencing. Most patients (n = 126) were HBeAg-negative (HBeAgNeg) with significantly lower levels of HBV-RNA, HBV-DNA and HBsAg compared to HBeAg-positive (HBeAgPos) patients (p < 0.05). HBV genotype D prevailed (61/68), and >95% had BCP/PC mutations. Escape mutations were identified in 22.6% (13/63). HBeAgNeg patients with low levels of HBsAg (log IU ≤ 3) were older and were characterized by undetectable plasma HBV-DNA and undetectable HBV-RNA but not undetectable HBV-DNA in PBMCs compared to those with high HBsAg levels. In >50% of the studied HBeAgNeg patients (66/126), the quantitation of HBsAg and HBV-RNA may impact clinical decisions. In conclusion, the combined assessment of classical and novel serum biomarkers, especially in HBeAgNeg patients, which is the largest group of CHB patients in many regions, may assist in clinical decisions. Prospective studies are required to determine the real-time additive clinical advantage of these biomarkers.