A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting.

Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ∼10 samples per experiment without substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to vertically, and (ii) a 6 mm thick gel is used, with 2 mm most relevant for membrane transfer (vs ∼1 mm typical). Results demonstrate both wet and semi-dry transfer are compatible with this gel thickness. The major tradeoff is reduced molecular weight resolution, due primarily to less available migration distance per sample. We demonstrate proof-of-principle using gels loaded with molecular weight ladder, recombinant protein, and cell lysates. We expect the 96-well western blot will increase reproducibility, efficiency, and capacity for biological characterization relative to established western blots.

Novel extended-release transdermal formulations of the psychedelic N,N-dimethyltryptamine (DMT).

There is considerable evidence from the literature that psychedelics, such as N,N-dimethyltryptamine (DMT), are safe and effective treatments for depression. However, clinical administration to induce psychedelic effects and expensive psychotherapy-assisted treatments likely limit accessibility to the average patient. There is emerging evidence that DMT promotes positive behavioral changes in vivo at sub-hallucinogenic dosages, and depending on the target indication, subjecting patients to high, bolus dosages may not be necessary. Due to rapid metabolic degradation, achieving target levels of DMT in subjects is difficult, requiring IV administration, which poses risks to patients during the intense hallucinogenic and subjective drug effects. The chemical and physical properties of DMT make it an excellent candidate for non-invasive, transdermal delivery platforms. This paper outlines the formulation development, in vitro, and in vivo testing of transdermal drug-in-adhesive DMT patches using various adhesives and permeation enhancers. In vivo behavioral and pharmacokinetic studies were performed with lead patch formulation (F5) in male and female Swiss Webster mice, and resulting DMT levels in plasma and brain samples were quantified using LC/MS/MS. Notable differences were seen in female versus male mice during IV administration; however, transdermal administration provided consistent, extended drug release at a non-hallucinogenic dose. The IV half-life of DMT was extended by 20-fold with administration of the transdermal delivery system at sub-hallucinogenic plasma concentrations not exceeding 60 ng/mL. Results of a translational head twitch assay (a surrogate for hallucinogenic effects in non-human organisms) were consistent with absence of hallucinations at low plasma levels achieved with our TDDS. Despite the reported low bioavailability of DMT, the non-invasive transdermal DMT patch F5 afforded an impressive 77 % bioavailability compared to IV at two dosages. This unique transdermal delivery option has the potential to provide an out-patient treatment option for ailments not requiring higher, bolus doses and is especially intriguing for therapeutic indications requiring non-hallucinogenic alternatives.

An effective COVID-19 vaccine hesitancy intervention focused on the relative risks of vaccination and infection.

We designed the Relative Risk Tool (RRT) to help people assess the relative risks associated with COVID-19 vaccination and infection. In May 2022 (N = 400) and November 2022 (N = 615), U.S. residents participated in a survey that included questions about the risks of vaccination and infection. In both cohorts, we found an association between relative risk perception and vaccine hesitancy. Participants in the May cohort were randomly assigned an intervention: to see information from the RRT or the Centers for Disease Control and Prevention (CDC). After the intervention, participants answered the same questions about risk perception and vaccination intent again. The RRT was more effective than the CDC at changing risk perception and increasing vaccination intent. In November, the survey structure was the same, but the RRT was the only intervention included, and we confirmed that the RRT was effective at changing opinions in this new sample. Importantly, the RRT provided accurate information about the risks of serious adverse outcomes to vaccination and still increased vaccination intent. Our work suggests that the RRT helps people assess relative risk, which can in turn help empower them to make informed decisions and ultimately reduce vaccine hesitancy.

CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs.

Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

Pharmacokinetic analysis of acute and dietary exposure to piperonyl butoxide in the mouse.

Piperonyl butoxide (PBO) is a popular insecticide synergist present in thousands of commercial, agricultural, and household products. PBO inhibits cytochrome P450 activity, impairing the ability of insects to detoxify insecticides. PBO was recently discovered to also inhibit Sonic hedgehog signaling, a pathway required for embryonic development, and rodent studies have demonstrated the potential for in utero PBO exposure to cause structural malformations of the brain, face, and limbs, or more subtle neurodevelopmental abnormalities. The current understanding of the pharmacokinetics of PBO in mice is limited, particularly with respect to dosing paradigms associated with developmental toxicity. To establish a pharmacokinetic (PK) model for oral exposure, PBO was administered to female C57BL/6J mice acutely by oral gavage (22-1800 mg/kg) or via diet (0.09 % PBO in chow). Serum and adipose samples were collected, and PBO concentrations were determined by HPLC-MS/MS. The serum concentrations of PBO were best fit by a linear one-compartment model. PBO concentrations in visceral adipose tissue greatly exceeded those in serum. PBO concentrations in both serum and adipose tissue decreased quickly after cessation of dietary exposure. The elimination half-life of PBO in the mouse after gavage dosing was 6.5 h (90 % CI 4.7-9.5 h), and systemic oral clearance was 83.3 ± 20.5 mL/h. The bioavailability of PBO in chow was 41 % that of PBO delivered in olive oil by gavage. Establishment of this PK model provides a foundation for relating PBO concentrations that cause developmental toxicity in the rodent models to Sonic hedgehog signaling pathway inhibition.

In vivo validation of psilacetin as a prodrug yielding modestly lower peripheral psilocin exposure than psilocybin.

Introduction: The use of the psychedelic compound psilocybin in conjunction with psychotherapy has shown promising results in the treatment of psychiatric disorders, though the underlying mechanisms supporting these effects remain unclear. Psilocybin is a Schedule I substance that is dephosphorylated in vivo to form an active metabolite, psilocin. Psilacetin, also known as O-acetylpsilocin or 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT), is an unscheduled compound that has long been suggested as an alternative psilocin prodrug, though direct in vivo support for this hypothesis has thus far been lacking. Methods: This study employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess the time-course and plasma concentrations of psilocin following the intraperitoneal (IP) administration of psilacetin fumarate or psilocybin to male and female C57Bl6/J mice. Results: Direct comparisons of the time courses for psilocin exposure arising from psilocybin and psilacetin found that psilocybin led to 10-25% higher psilocin concentrations than psilacetin at 15-min post-injection. The half-life of psilocin remained approximately 30 min, irrespective of whether it came from psilocybin or psilacetin. Overall, the relative amount of psilocin exposure from psilacetin fumarate was found to be approximately 70% of that from psilocybin. Discussion: These findings provide the first direct support for the long-standing assumption in the field that psilacetin functions as a prodrug for psilocin in vivo. In addition, these results indicate that psilacetin fumarate results in lower peripheral psilocin exposure than psilocybin when dosed on an equimolar basis. Thoughtful substitution of psilocybin with psilacetin fumarate appears to be a viable approach for conducting mechanistic psychedelic research in C57Bl6/J mice.

Mesowestern Blot: Simultaneous Analysis of Hundreds of Submicroliter Lysates.

Western blotting is a widely used technique for molecular-weight-resolved analysis of proteins and their posttranslational modifications, but high-throughput implementations of the standard slab gel arrangement are scarce. The previously developed Microwestern requires a piezoelectric pipetting instrument, which is not available for many labs. Here, we report the Mesowestern blot, which uses a 3D-printable gel casting mold to enable high-throughput Western blotting without piezoelectric pipetting and is compatible with the standard sample preparation and small (∼1 µL) sample sizes. The main tradeoffs are reduced molecular weight resolution and higher sample-to-sample CV, making it suitable for qualitative screening applications. The casted polyacrylamide gel contains 336, ∼0.5 µL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution profiles. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder and cell lysate (RIPA buffer) demonstrate that the protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semidry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is at least 32-fold, and at least ∼500 attomols of ß-actin can be detected (∼29 ng of total protein from mammalian cell lysates: ∼100-300 cells). Because the gel mold is 3D-printable, users with access to additive manufacturing cores have significant design freedom for custom layouts. We expect that the technique could be easily adopted by any typical cell and molecular biology laboratory already performing Western blots.

Under-Oil Autonomously Regulated Oxygen Microenvironments: A Goldilocks Principle-Based Approach for Microscale Cell Culture.

Oxygen levels in vivo are autonomously regulated by a supply-demand balance, which can be altered in disease states. However, the oxygen levels of in vitro cell culture systems, particularly microscale cell culture, are typically dominated by either supply or demand. Further, the oxygen microenvironment in these systems is rarely monitored or reported. Here, a method to establish and dynamically monitor autonomously regulated oxygen microenvironments (AROM) using an oil overlay in an open microscale cell culture system is presented. Using this method, the oxygen microenvironment is dynamically regulated via the supply-demand balance of the system. Numerical simulation and experimental validation of oxygen transport within multi-liquid-phase, microscale culture systems involving a variety of cell types, including mammalian, fungal, and bacterial cells are presented. Finally, AROM is applied to establish a coculture between cells with disparate oxygen demands-primary intestinal epithelial cells (oxygen consuming) and Bacteroides uniformis (an anaerobic species prevalent in the human gut).

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6; that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3; and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis.IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with the MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

A randomized, double-blind, dose-ranging, pilot trial of piperine with resveratrol on the effects on serum levels of resveratrol.

Resveratrol (3,4,5-trihydroxystilbene) is a naturally occurring phytoalexin with purported health-promoting effects, but with limited oral bioavailability. Our prior murine modeling research observed enhanced resveratrol bioavailability with piperine co-administration. In this study, single-dose pharmacokinetics of resveratrol with or without piperine and the associated toxicities were studied on a cohort of healthy volunteers. We performed a double-blind, randomized, three-arm pilot study. Participants were randomized to receive a single dose of resveratrol 2.5 g, with piperine in 0 mg, 5 mg, or 25 mg dose. An improved liquid chromatography/mass spectrometry assay was used to determine serum levels of resveratrol and resveratrol-glucuronide. Baseline through 24 h post-study drug serum analyses were performed and adverse events were followed for 30 days. Twenty-four participants were enroled. No significant relationship between dose and pharmacokinetic values were found. In the sex stratified analysis, Cmax for resveratrol in women showed a trend (P = 0.057) toward an increase with piperine. Pharmacokinetic values for resveratrol were: Cmax - 18.5 ± 16 ng/mL resveratrol alone, 29 ± 29 resveratrol + 5 mg piperine, 16 ± 13 resveratrol + 25 mg piperine; area under the concentration × time curve - 1270 ± 852 ng/h/mL resveratrol alone, 2083 ± 2284 resveratrol + 5 mg piperine, 1132 ± 222 resveratrol + 25 mg piperine. All subjects tolerated their protocol therapy with minimal to no toxicity and no evidence of differences between the three groups. The co-administration of resveratrol with piperine at 5 and 25 mg doses did not sufficiently alter the pharmacokinetics of resveratrol or resveratrol-glucuronide to demonstrate the significant enhancement observed in murine modeling.

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit. HIGHLIGHTS: · ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species· Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways· Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function· Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection.

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit. Highlights{middle dot} ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species {middle dot} Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways {middle dot} Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function {middle dot} Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-ß signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.

Trehalose Synthesis Contributes to Osmotic Stress Tolerance and Virulence of the Bacterial Wilt Pathogen Ralstonia solanacearum.

The xylem-dwelling plant pathogen Ralstonia solanacearum changes the chemical composition of host xylem sap during bacterial wilt disease. The disaccharide trehalose, implicated in stress tolerance across all kingdoms of life, is enriched in sap from R. solanacearum-infected tomato plants. Trehalose in xylem sap could be synthesized by the bacterium, the plant, or both. To investigate the source and role of trehalose metabolism during wilt disease, we evaluated the effects of deleting the three trehalose synthesis pathways in the pathogen: TreYZ, TreS, and OtsAB, as well as its sole trehalase, TreA. A quadruple treY/treS/otsA/treA mutant produced 30-fold less intracellular trehalose than the wild-type strain missing the trehalase enzyme. This trehalose-nonproducing mutant had reduced tolerance to osmotic stress, which the bacterium likely experiences in plant xylem vessels. Following naturalistic soil-soak inoculation of tomato plants, this triple mutant did not cause disease as well as wild-type R. solanacearum. Further, the wild-type strain out-competed the trehalose-nonproducing mutant by over 600-fold when tomato plants were coinoculated with both strains, showing that trehalose biosynthesis helps R. solanacearum overcome environmental stresses during infection. An otsA (trehalose-6-phosphate synthase) single mutant behaved similarly to ΔtreY/treS/otsA in all experimental settings, suggesting that the OtsAB pathway is the dominant trehalose synthesis pathway in R. solanacearum.

Single centre experience of bilateral gracilis flap perineal reconstruction following extra-levator abdominoperineal excision.

AIM: A variety of tissue flaps have been described for the closure of perineal wounds following abdominoperineal excision of the rectum (APE) or exenteration for locally advanced/recurrent rectal cancer and salvage surgery for anal cancer. The aim of this study was to demonstrate the utility of the bilateral pedicled gracilis muscle flaps (BPGMFs) as a reconstruction option in these patients. This is of particular benefit when using a laparoscopic approach for the abdominal component of the operation, avoiding disruption of the abdominal wall and risk of herniation with other reconstruction options, e.g. vertical rectus abdominis myocutaneous flaps. METHOD: This is a retrospective single centre case series of patients who underwent reconstruction of perineal defects using BPGMFs using a novel weave technique, from January 2008 to August 2017. RESULTS: There were 25 patients (16 female), with a median follow-up of 19 months (3-102). The indications for BPGMFs were cancer resection (21) and perineal hernia (4). The median length of stay was 14 days (6-60). All-cause mortality was 36% within the follow-up period. A healed perineal wound was achieved in 72% of patients within 30 days (84% of patients received neoadjuvant chemoradiotherapy). The overall donor site complication rate was 20% (including infection, dehiscence, numbness, haematoma and seroma) and 28% for the perineal site (including infection, dehiscence and prolapse). CONCLUSIONS: BPGMFs provide an important option for reconstruction of the perineum particularly with a minimally invasive approach or with two stomas.

Low-fat yogurt consumption reduces biomarkers of chronic inflammation and inhibits markers of endotoxin exposure in healthy premenopausal women: a randomised controlled trial.

The anti-inflammatory mechanisms of low-fat dairy product consumption are largely unknown. The objective of this study was to determine whether low-fat yogurt reduces biomarkers of chronic inflammation and endotoxin exposure in women. Premenopausal women (BMI 18·5-27 and 30-40 kg/m2) were randomised to consume 339 g of low-fat yogurt (yogurt non-obese (YN); yogurt obese (YO)) or 324 g of soya pudding (control non-obese; control obese (CO)) daily for 9 weeks (n 30/group). Fasting blood samples were analysed for IL-6, TNF-α/soluble TNF II (sTNF-RII), high-sensitivity C-reactive protein, 2-arachidonoyl glycerol, anandamide, monocyte gene expression, soluble CD14 (sCD14), lipopolysaccharide (LPS), LPS binding protein (LBP), IgM endotoxin-core antibody (IgM EndoCAb), and zonulin. BMI, waist circumference and blood pressure were also determined. After 9-week yogurt consumption, YO and YN had decreased TNF-α/sTNFR-RII. Yogurt consumption increased plasma IgM EndoCAb regardless of obesity status. sCD14 was not affected by diet, but LBP/sCD14 was lowered by yogurt consumption in both YN and YO. Yogurt intervention increased plasma 2-arachidonoylglycerol in YO but not YN. YO peripheral blood mononuclear cells expression of NF-κB inhibitor α and transforming growth factor ß1 increased relative to CO at 9 weeks. Other biomarkers were unchanged by diet. CO and YO gained approximately 0·9 kg in body weight. YO had 3·6 % lower diastolic blood pressure at week 3. Low-fat yogurt for 9 weeks reduced biomarkers of chronic inflammation and endotoxin exposure in premenopausal women compared with a non-dairy control food. This trial was registered as NCT01686204.

Molecular signatures of sanguinarine in human pancreatic cancer cells: A large scale label-free comparative proteomics approach.

Pancreatic cancer remains one of the most lethal of all human malignancies with its incidence nearly equaling its mortality rate. Therefore, it's crucial to identify newer mechanism-based agents and targets to effectively manage pancreatic cancer. Plant-derived agents/drugs have historically been useful in cancer therapeutics. Sanguinarine is a plant alkaloid with anti-proliferative effects against cancers, including pancreatic cancer. This study was designed to determine the mechanism of sanguinarine's effects in pancreatic cancer with a hope to obtain useful information to improve the therapeutic options for the management of this neoplasm. We employed a quantitative proteomics approach to define the mechanism of sanguinarine's effects in human pancreatic cancer cells. Proteins from control and sanguinarine-treated pancreatic cancer cells were digested with trypsin, run by nano-LC/MS/MS, and identified with the help of Swiss-Prot database. Results from replicate injections were processed with the SIEVE software to identify proteins with differential expression. We identified 37 differentially expressed proteins (from a total of 3107), which are known to be involved in variety of cellular processes. Four of these proteins (IL33, CUL5, GPS1 and DUSP4) appear to occupy regulatory nodes in key pathways. Further validation by qRT-PCR and immunoblot analyses demonstrated that the dual specificity phosphatase-4 (DUSP4) was significantly upregulated by sanguinarine in BxPC-3 and MIA PaCa-2 cells. Sanguinarine treatment also caused down-regulation of HIF1α and PCNA, and increased cleavage of PARP and Caspase-7. Taken together, sanguinarine appears to have pleotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness of sanguinarine against pancreatic cancer.