Multimodal decoding of human liver regeneration.

The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.

TiF4-catalyzed direct amidation of carboxylic acids and amino acids with amines.

Unlike other metal fluorides, catalytic titanium tetrafluoride enhances the direct amidation of aromatic and aliphatic carboxylic acids and N-protected amino acids in refluxing toluene. While aromatic acids were converted to amides with 10 mol% of the catalyst within 24 h, aliphatic acids underwent a faster reaction (12 h), with lower catalyst loading (5 mol%). This protocol is equally efficient with alkyl and aryl amines providing a variety of carboxamides and peptides in 60-99% yields.

Borane-Pyridine: An Efficient Catalyst for Direct Amidation.

Borane-pyridine acts as an efficient (5 mol%) liquid catalyst, providing improved solubility for the direct amidation of a wide range of aromatic and aliphatic carboxylic acids and amines to form secondary and tertiary carboxamides. Tolerance of potentially incompatible halo, nitro, and alkene functionalities has been demonstrated.

One-Pot, Tandem Reductive Amination/Alkylation-Cycloamidation for Lactam Synthesis from Keto or Amino Acids.

Monotrifluoroacetoxyborane-amines, prepared by treating borane-amines with trifluoroacetic acid, have been shown to be efficient reagents for a one-pot, tandem reductive amination/alkylation-cycloamidation of keto or amino acids to achieve the synthesis of 5-aryl or 5-methyl pyrrolidin-2-ones and 6-aryl or 6-methyl piperidin-2-ones.

Dehydroborylation of Terminal Alkynes Using Lithium Aminoborohydrides.

Dehydrogenative borylation of terminal alkynes has recently emerged as an atom-economical one-step alternative to traditional alkyne borylation methodologies. Using lithium aminoborohydrides, formed in situ from the corresponding amine-boranes and n-butyllithium, a variety of aromatic and aliphatic terminal alkyne substrates were successfully borylated in high yield. The potential to form mono-, di-, and tri-B-alkynylated products has been shown, though the mono-product is primarily generated using the presented condition. The reaction has been demonstrated at large (up to 50 mmol) scale, and the products are stable to column chromatography as well as acidic and basic aqueous conditions. Alternately, the dehydroborylation can be achieved by treating alkynyllithiums with amine-boranes. In that respect, aldehydes can act as starting materials by conversion to the 1,1-dibromoolefin and in situ rearrangement to the lithium acetylide.

Titanium-Mediated Reduction of Carboxamides to Amines with Borane-Ammonia.

In this study, the successful titanium tetrachloride-catalyzed reduction of aldehydes, ketones, carboxylic acids, and nitriles with borane-ammonia was extended to the reduction (deoxygenation) of a variety of aromatic and aliphatic pri-, sec- and tert-carboxamides, by changing the stoichiometry of the catalyst and reductant. The corresponding amines were isolated in good to excellent yields, following a simple acid-base workup.

TiCl4-Catalyzed Hydroboration of Ketones with Ammonia Borane.

Investigation of a variety of Lewis acids for the hydroboration-hydrolysis (reduction) of ketones with amine-boranes has revealed that catalytic (10 mol %) titanium tetrachloride (TiCl4) in diethyl ether at room temperature immensely accelerates the reaction of ammonia borane. The product alcohols are produced in good to excellent yields within 30 min, even with ketones which typically requires 24 h or longer to reduce under uncatalyzed conditions. Several potentially reactive functionalities are tolerated, and substituted cycloalkanones are reduced diastereoselectively to the thermodynamic product. A deuterium labeling study and 11B NMR analysis of the reaction have been performed to verify the proposed hydroboration mechanism.

Room Temperature Reduction of Titanium Tetrachloride-Activated Nitriles to Primary Amines with Ammonia-Borane.

The reduction of a variety of aromatic and aliphatic nitriles, activated by a molar equivalent of titanium tetrachloride, has been achieved at room temperature using ammonia borane as a safe reductant. The corresponding methanamines were isolated in good to excellent yields following a simple acid-base workup.

Trimethyl Borate-Catalyzed, Solvent-Free Reductive Amination.

Solvent-free reductive amination of aldehydes and ketones with aliphatic and aromatic amines in high-to-excellent yields has been achieved with sub-stoichiometric trimethyl borate as promoter and ammonia borane as reductant.

Development and validation of a structured feedback questionnaire from postgraduates on various elements of postgraduate medical curriculum.

BACKGROUND: Medical Council of India, introduced the Post Graduate (PG) curriculum as 'Competency Based Medical Education' (CBME). Feedback from the end users is a vital step in curriculum evaluation. Therefore, the primary objective of this study was to develop and validate a Structured Feedback Questionnaire (SFQ) for postgraduates, encompassing all the components of the PG-CBME curriculum. METHODS: SFQ was developed with 23 Likert based questions and four open ended questions. Content validation was done by Lawshe method. After getting institutional ethics clearance and informed consent, SFQ was administered to 121 final year PGs (response rate 100%). We performed Principal component analysis (PCA), Structural equation modeling (SEM), Chi squared test (χ2/df); goodness-of-fit index (GFI); adjusted GFI; comparative fit index (CFI) and root mean square error of approximation (RMSEA). Cronbach's alpha was done for estimating the internal consistency. RESULTS: The validation resulted in a three-factor model comprising of "curriculum" (42.1%), "assessment" (28%), and "support" (18.5%). Chi squared test (χ2/df ratio) < 2, CFI (0.78), GFI (0.72) and RMSEA (0.09) indicated superior goodness of fit for the three-factor model for the sample data. All the extracted factors had good internal consistency of ≥0.9. CONCLUSION: We believe that this 23 item SFQ is a valid and reliable tool which can be utilized for curriculum evaluation and thereby formulating recommendations to modify the existing curriculum wherever required, facilitating enriched program outcomes.

Female ageing affects the DNA repair capacity of oocytes in IVF using a controlled model of sperm DNA damage in mice.

STUDY QUESTION: Does female ageing have a negative effect on the DNA repair capacity of oocytes fertilised by spermatozoa with controlled levels of DNA damage? SUMMARY ANSWER: Compared to oocytes from younger females, oocytes from older females have a reduced capacity to repair damaged DNA introduced by spermatozoa. WHAT IS KNOWN ALREADY: The reproductive lifespan in women declines with age predominantly due to poor oocyte quality. This leads to decreased reproductive outcomes for older women undergoing assisted reproductive technology (ART) treatments, compared to young women. Ageing and oocyte quality have been clearly associated with aneuploidy, but the range of factors that influence this change in oocyte quality with age remains unclear. The DNA repair activity prior to embryonic genomic activation is considered to be of maternal origin, with maternal transcripts and proteins controlling DNA integrity. With increasing maternal age, the number of mRNAs stored in oocytes decreases. This could result in diminished efficiency of DNA repair and/or negative effects on embryo development, especially in the presence of DNA damage. STUDY DESIGN, SIZE, DURATION: Oocytes from two age groups of 30 super-ovulated female mice (young: 5-8 weeks old, n = 15; old: 42-45 weeks old, n = 15) were inseminated with sperm from five males with three different controlled DNA damage levels; control: ≤10%, 1 Gray (Gy): 11-30%, and 30 Gy: >30%. Inseminated oocytes (young: 125, old: 78) were assessed for the formation of zygotes (per oocyte) and blastocysts (per zygote). Five replicates of five germinal vesicles (GVs) and five MII oocytes from each age group were analysed for gene expression. The DNA damage response (DDR) was assessed in a minimum of three IVF replicates in control and 1 Gy zygotes and two-cell embryos using γH2AX labelling. PARTICIPANTS/MATERIALS, SETTING, METHODS: Swim-up sperm samples from the cauda epididymidis of C57BL6 mice were divided into control (no irradiation) and 1- and 30-Gy groups. Treated spermatozoa were irradiated at 1 and 30 Gy, respectively, using a linear accelerator Varian 21iX. Following irradiation, samples were used for DNA damage assessment (Halomax) and for insemination. Presumed zygotes were cultured in a time-lapse incubator (MIRI, ESCO). Gene expression of 91 DNA repair genes was assessed using the Fluidigm Biomark HD system. The DNA damage response in zygotes (6-8 h post-fertilisation) and two-cell embryos (22-24 h post-fertilisation) was assessed by immunocytochemical analysis of γH2AX using confocal microscopy (Olympus FV1200) and 3D volumetric analysis using IMARIS software. MAIN RESULTS AND THE ROLE OF CHANCE: The average sperm DNA damage for the three groups was statistically different (control: 6.1%, 1 Gy: 16.1%, 30 Gy: 53.1%, P < 0.0001), but there were no significant differences in fertilisation rates after IVF within or between the two age groups [(young; control: 86.79%, 1 Gy: 82.75%, 30 Gy: 76.74%) (old; control: 93.1%, 1 Gy: 70.37%, 30 Gy: 68.18%) Fisher's exact]. However, blastocyst rates were significantly different (P < 0.0001) among the groups [(young; control: 86.95%, 1 Gy: 33.33%, 30 Gy: 0.0%) (old; control: 70.37%, 1 Gy: 0.0%, 30 Gy: 0.0%)]. Between the age groups, 1-Gy samples showed a significant decrease in the blastocyst rate in old females compared to young females (P = 0.0166). Gene expression analysis revealed a decrease in relative expression of 21 DNA repair genes in old GV oocytes compared to young GV oocytes (P < 0.05), and similarly, old MII oocytes showed 23 genes with reduced expression compared to young MII oocytes (P < 0.05). The number of genes with decreased expression in older GV and MII oocytes significantly affected pathways such as double strand break (GV: 5; MII: 6), nucleotide excision repair (GV: 8; MII: 5) and DNA damage response (GV: 4; MII: 8). There was a decreased DDR in zygotes and in two-cell embryos from old females compared to young regardless of sperm treatment (P < 0.05). The decrease in DNA repair gene expression of oocytes and decreased DDR in embryos derived from older females suggests that ageing results in a diminished DNA repair capacity. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ionising radiation was used only for experimental purposes, aiming at controlled levels of sperm DNA damage; however, it can also damage spermatozoa proteins. The female age groups selected in mice were intended to model effects in young and old women, but clinical studies are required to demonstrate a similar effect. WIDER IMPLICATIONS OF THE FINDINGS: Fertilisation can occur with sperm populations with medium and high DNA damage, but subsequent embryo growth is affected to a greater extent with aging females, supporting the theory that oocyte DNA repair capacity decreases with age. Assessment of the oocyte DNA repair capacity may be a useful diagnostic tool for infertile couples. STUDY FUNDING/COMPETING INTEREST(S): Funded by the Education Program in Reproduction and Development, Department of Obstetrics and Gynaecology, Monash University. None of the authors has any conflict of interest to report.

ß,γ-Diaryl α-methylene-γ-butyrolactones as potent antibacterials against methicillin-resistant Staphylococcus aureus.

A selected series of racemic α-methylene-γ-butyrolactones (AMGBL) synthesized via allylboration or allylindation reactions were screened against methicillin-resistant Staphylococcus aureus (MRSA) USA300. Unlike natural AMGBLs, such as parthenolide, synthetic analogs bearing aryl moieties at the ß- and γ-positions are potent against MRSA. The most potent molecules were comparable to vancomycin and linezolid, the drugs of the last resort for MRSA infections, in their effectiveness with minimum inhibitory concentrations (MICs) ranging from 3.0 to 5.2 µM. These lactones also exhibited potent antibacterial activity against other clinically important multidrug-resistant Gram-positive bacteria (except enterococci), while also showing high tolerability to mammalian cells. Several of these molecules surpassed vancomycin in their rapid killing of the high MRSA inoculum (2 h vs 12 h) in a standard time-kill kinetics assay, with compounds 1l and 1m significantly reducing the intracellular burden of MRSA by about 98-99%, at low concentrations. Additionally, the compounds surpassed vancomycin in inhibiting staphylococcal protease production, indicating that synthetic methylene lactones warrant further investigations as promising anti-MRSA candidates.

Safety in moderate-to-severe plaque psoriasis patients with latent tuberculosis treated with guselkumab and anti-tuberculosis treatments concomitantly: results from pooled phase 3 VOYAGE 1 & VOYAGE 2 trials.

BACKGROUND: Patients treated with tumour necrosis factor (TNF) inhibitors are at risk of new-onset tuberculosis (TB) or reactivation of latent tuberculosis infection (LTBI). Association between TB/LTBI and interleukin (IL)-23 inhibitors for psoriasis is unclear. Patients with LTBI typically initiate LTBI therapy before receiving biologics. OBJECTIVES: Safety in moderate-to-severe psoriasis patients with LTBI treated with guselkumab (IL-23 inhibitor) and LTBI treatment was evaluated. METHODS: In the VOYAGE 1 & VOYAGE 2 studies, patients screened for LTBI were randomized to guselkumab, placebo, or adalimumab (TNF inhibitor) at baseline. Placebo â†’ guselkumab crossover occurred at week 16 and adalimumab â†’ guselkumab at week 52 (VOYAGE 1), or at week 28 or later (VOYAGE 2). Incidence of active TB, adverse events (AEs), serious AEs (SAEs), and markedly abnormal liver function tests [alanine aminotransferase test (ALT); aspartate aminotransferase test (AST)] were evaluated using pooled data through week 100 in guselkumab-treated patients receiving and not receiving LTBI treatment. RESULTS: At baseline, 130 randomized patients (guselkumab: n = 69; adalimumab: n = 36; placebo: n = 25) tested positive for LTBI and received concomitant LTBI treatments (LTBI+). No active TB was reported among guselkumab-treated patients without LTBI (LTBI-) through week 100. Two cases of active TB occurred in LTBI- patients treated with adalimumab. Through week 16, across all treatment groups, greater proportions of LTBI+ patients reported ALT and AST elevations compared with LTBI- patients. Through week 100, proportions of patients experiencing AEs and SAEs were comparable between LTBI+ and LTBI- patients. CONCLUSIONS: No cases of active TB, including reactivation of LTBI, were reported in patients with or without LTBI treated with guselkumab through up to 2 years. LTBI treatment was effective across all treatment groups in preventing reactivation of LTBI. Long-term treatment with guselkumab was generally well-tolerated through up to 2 years in patients receiving LTBI medications.

Factors influencing the cytotoxicity of α-methylene-γ-hydroxy esters against pancreatic cancer.

A systematic study to identify the factors influencing the cytotoxicity of α-methylene-γ-hydroxy esters against three pancreatic cancer cell lines (Panc-1, MIA-PaCa-2, and BxPC-3) has established that, in addition to Michael acceptor abilities, the possibility to lactonize to α-methylene-γ-butyrolactones is as important. The substitution pattern and the number of carbons between the hydroxy and ester moieties also influence the bio-activity.

Open-Flask Synthesis of Amine-Boranes via Tandem Amine-Ammonium Salt Equilibration-Metathesis.

An amine-ammonium salt equilibration-metathesis sequence provides high-purity amine-boranes in excellent yields from sodium borohydride in refluxing reagent-grade tetrahydrofuran in an open flask.

Development and utility of the FDA 'GutProbe' DNA microarray for identification, genotyping and metagenomic analysis of commercially available probiotics.

AIM: Lactic acid bacteria are beneficial microbes added to many food products and dietary supplements for their purported health benefits. Proper identification of bacteria is important to assess safety as well as proper product labelling. A custom microarray (FDA GutProbe) was developed to verify accurate labelling in commercial dietary supplements. METHODS AND RESULTS: Strain-specific attribution was achieved with GutProbe array which contains genes from the most commonly found species in probiotic supplements and food ingredients. Applied utility of the array was assessed with direct from product DNA hybridization to determine (i) if identification of multiple strains in one sample can be conducted and (ii) if any lot-to-lot variations exist with eight probiotics found on the US market. CONCLUSIONS: GutProbe is a useful tool in identifying a mixture of microbials in probiotics and did reveal some product variations. In addition, the array is able to identify lot-to-lot differences in these products. These strain level attribution may be useful for routine monitoring of batch variation as part of a 'Good Manufacturing Practices' process. SIGNIFICANCE AND IMPACT OF THE STUDY: The FDA GutProbe is an efficient and reliable platform to identify the presence of microbial ingredients and determining microbe differences in dietary supplements. The GutProbe is a fast, rapid method for direct community profiling or food matrix sampling.

UV photofragmentation and IR spectroscopy of cold, G-type ß-O-4 and ß-ß dilignol-alkali metal complexes: structure and linkage-dependent photofragmentation.

Ultraviolet photofragmentation spectroscopy and infrared spectroscopy were performed on two prototypical guaiacyl (G)-type dilignols containing ß-O-4 and ß-ß linkages, complexed with either lithium or sodium cations. The complexes were generated by nanoelectrospray ionization, introduced into a multistage mass spectrometer, and subsequently cooled in a 22-pole cold ion trap to T ≈ 10 K. A combination of UV photofragment spectroscopy and IR-UV double resonance spectroscopy was used to characterize the preferred mode of binding of the alkali metal cations and the structural changes so induced. Based on a combination of spectral evidence provided by the UV and IR spectra, the Li(+) and Na(+) cations are deduced to preferably bind to both dilignols via their linkages, which constitute unique, oxygen-rich binding pockets for the cations. The UV spectra reflect this binding motif in their extensive Franck-Condon activity involving low-frequency puckering motions of the linkages in response to electronic excitation. In the pinoresinol•Li(+)/Na(+) complexes involving the ß-ß linkage, the spectra also showed an inherent spectral broadening. The photofragment mass spectra are unique for each dilignol•Li(+)/Na(+) complex, many of which are also complementary to those produced by collision-induced dissociation (CID), indicating the presence of unique excited state processes that direct the fragmentation. These results suggest the potential for site-selective fragmentation and for uncovering fragmentation pathways only accessed by resonant UV excitation of cold lignin ions.

Amine-boranes: green hypergolic fuels with consistently low ignition delays.

Complexation of amines with borane converts them to hypergols or decreases their ignition delays (IDs) multifold (with white fuming nitric acid as the oxidant). With consistently low IDs, amine-boranes represent a class of compounds that can be promising alternatives to toxic hydrazine and its derivatives as propellants. A structure-hypergolicity relationship study reveals the necessary features for the low ID.

Outcome of partial internal biliary diversion for intractable pruritus in children with cholestatic liver disease.

PURPOSE: Children with cholestatic disorders have undergone liver transplantation for intractable pruritus unresponsive to medical therapy even in the absence of liver failure. Biliary diversion procedures interrupt the entero-hepatic circulation of bile acids allowing them to be excreted in the feces thereby lowering the total bile acid pool. We evaluated the outcome of partial internal biliary diversion (PIBD) in children with intractable pruritus from inherited cholestatic disorders. METHODS: The records of children who underwent PIBD over a 4-year period were reviewed for etiology of liver disease, demographic data, preoperative and postoperative biochemical profile and improvement of pruritus. Standard statistical methods were used for analysis. RESULTS: Of the 12 children, 10 had progressive familial intrahepatic cholestasis (PFIC) and 2 had Alagille syndrome (AS). PIBD was done using an isolated jejunal loop as a conduit from gall bladder to mid ascending colon. Median period of follow up was 30 months. Pruritus resolved in nine children with significant reduction of serum bile acids (P < 0.02). CONCLUSION: To our knowledge, this is the largest reported series of children with PIBD. PIBD is a safe, well-tolerated and effective alternative to liver transplant in children with PFIC and AS who have intractable pruritus in the absence of synthetic liver failure.