Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Small Peptides Targeting BACE-1, AChE, and A-ß Reversing Scopolamine-Induced Memory Impairment: A Multitarget Approach against Alzheimer's Disease.

Based on the biochemical understanding of Alzheimer's disease, here, we report the design, synthesis, and biological screening of a series of compounds against this neuro-disorder. Adopting the multitarget approach, the catalytic processes of BACE-1 and AChE were targeted, and thereby, compounds 15, 22, 25, 26, 27, and 30 were identified with IC50 in the submicromolar range against these two enzymes. Further, compounds 15 and 25 displayed more than 50% inhibition of ß-amyloid aggregation. Implying their physiological use, the compounds exhibited appreciable biological membrane permeability as observed through the parallel artificial membrane permeability experiment. Supporting these results, treatment of the mice with the test compounds reversed their scopolamine-affected memory impairment, where the highest healing effect was seen in the case of compound 25. Overall, the combination of molecular modeling and experimental studies provided highly effective molecules against Alzheimer's disease.

Design and Discovery of Novel NLRP3 Inhibitors and PET Imaging Radiotracers Based on a 1,2,3-Triazole-Bearing Scaffold.

The NOD-like receptor (NLR) family pyrin-domain-containing 3 (NLRP3) inflammasome, an essential component of the innate immune system, has been emerging as a viable drug target and a potential biomarker for human diseases. In our efforts to develop novel small molecule NLRP3 inhibitors, a 1-(5-chloro-2-methoxybenzyl)-4-phenyl-1H-1,2,3-triazole scaffold was designed via a rational approach based on our previous leads. Structure-activity relationship studies and biophysical studies identified a new lead compound 8 as a potent (IC50: 0.55 ± 0.16 µM), selective, and direct NLRP3 inhibitor. Positron emission tomography (PET) imaging studies of [11C]8 demonstrated its rapid and high brain uptake as well as fast washout in mice and rhesus macaque. Notably, plasma kinetic analysis of this radiotracer from the PET/magnetic resonance imaging studies in rhesus macaque suggested radiometabolic stability. Collectively, our data not only encourage further studies of this lead compound but also warrant further optimization to generate additional novel NLRP3 inhibitors and suitable central nervous system PET radioligands with translational promise.

Axitinib in patients with advanced/metastatic soft tissue sarcoma (Axi-STS): an open-label, multicentre, phase II trial in four histological strata.

BACKGROUND: Axitinib is an oral vascular endothelial growth factor receptor inhibitor with anti-tumour activity in renal, thyroid, and pancreatic cancer. METHODS: Axi-STS was a pathologically-stratified, non-randomised, open-label, multi-centre, phase II trial of continuous axitinib treatment in patients ≥16 years, performance status ≤2, with pathologically-confirmed advanced/metastatic soft tissue sarcoma (STS). Patients were recruited within four tumour strata, each analysed separately: angiosarcoma, leiomyosarcoma, synovial sarcoma, or other eligible STSs. The primary outcome was progression-free survival at 12 weeks (PFS12). A Simon's two-stage design with activity defined as PFS12 rate of 40% determined a sample size of 33 patients per strata. RESULTS: Between 31-August-2010 and 29-January-2016, 145 patients were recruited: 38 angiosarcoma, 37 leiomyosarcoma, 36 synovial sarcoma, and 34 other subtypes. PFS12 rate for each stratum analysed was 42% (95% lower confidence interval (LCI); 29), 45% (95% LCI; 32), 57% (95% LCI; 42), and 33% (95% LCI; 21), respectively. There were 74 serious adverse events including two treatment-related deaths of pulmonary haemorrhage and gastrointestinal bleeding. Fatigue and hypertension were the most common grade 3 adverse events. CONCLUSIONS: Axitinib showed clinical activity in all STS strata investigated. The adverse event profile was acceptable, supporting further investigation in phase III trials. CLINICAL TRIAL REGISTRATION: ISRCTN 60791336.

A patent review of NLRP3 inhibitors to treat autoimmune diseases.

INTRODUCTION: NOD-like receptor family pyrin domain containing 3 (NLRP3) can sense a plethora of exogenous and endogenous dangers. Upon activation, a multimeric protein complex, the NLRP3 inflammasome, is formed to initiate the innate immune responses. Emerging studies have implicated the pathophysiological roles of this protein complex in human disorders, highlighting that it represents a druggable target for therapeutics development. AREAS COVERED: The current review summarizes the functional facets of the NLRP3 inflammasome, its association with autoimmune diseases, and recent patents on the development of NLRP3 inhibitors. Literature search was conducted using SciFinder and Google Patents with the key word NLRP3 and NLRP3 inhibitors. EXPERT OPINION: Although significant advances have been made in understanding the NLRP3 inflammasome, more studies are still needed to elucidate the molecular mechanisms underlying its roles in autoimmune diseases. A number of NLRP3 inhibitors have been patented, however, none of them have been approved for clinical use. Due to the complex nature of the NLRP3 inflammasome, novel screening assays along with target engagement methods could benefit the drug discovery and clinical translation. In addition, clinical trials on NLRP3 inhibitors are still in their early stages, and continuous investigations are needed to fully assess their safety and effectiveness.

Improving neonatal health with family-centered, early postnatal care: A quasi-experimental study in India.

Despite the global decline, neonatal mortality rates (NMR) remain high in India. Family members are often responsible for the postpartum care of neonates and mothers. Yet, low health literacy and varied beliefs can lead to poor health outcomes. Postpartum education for family caregivers, may improve the adoption of evidence-based neonatal care and health outcomes. The Care Companion Program (CCP) is a hospital-based, pre-discharge health training session where nurses teach key healthy behaviors to mothers and family members, including skills and an opportunity to practice them in the hospital. We conducted a quasi-experimental study to assess the effect of the CCP sessions on mortality outcomes among families seeking care in 28 public tertiary facilities across 4 Indian states. Neonatal mortality outcomes were reported post-discharge, collected via phone surveys at four weeks postpartum, between October 2018 to February 2020. Risk ratios (RR), adjusting for hospital-level clustering, were calculated by comparing mortality rates before and after CCP implementation. A total of 46,428 families participated in the pre-intervention group and 87,305 in the post-intervention group; 76% of families completed the phone survey. Among the 33,599 newborns born before the CCP implementation, there were 1386 deaths (NMR: 41.3 deaths per 1000 live births). After the intervention began, there were 2021 deaths out of 60,078 newborns born (crude NMR: 33.6 deaths per 1000 live births, RR = 0.82, 95% CI: 0.76, 0.87; cluster-adjusted RR = 0.82, 95% CI: 0.71, 0.94). There may be a substantial benefit to family-centered education in the early postnatal period to reduce neonatal mortality.

Can training over phone calls help improve outcomes for COVID-19 positive patients under home isolation? An analysis of the COVID-19 Care Companion Program in Punjab, India.

Background: Phone-supported recovery of COVID-19 patients in home isolation could be an effective way of addressing COVID-19 in contexts with limited resources. The COVID-19 Care Companion Program (CCP) is one such intervention, designed to support patients and their caregivers in remote, evidence-based management of COVID-19 symptoms. Objective: To estimate the effect of providing phone-based training to COVID-19 patients and their caregivers on the likelihood of hospitalizations and mortality. Methods: A pragmatic randomized trial was conducted to assess the effect of a novel phone-based training program on COVID-19 home-isolated patient outcomes. The analysis compared the outcomes of death and hospitalizations in the teletraining intervention group (CCP) to those receiving standard of care (SoC). Results: Logistic regression models adjusted for age, gender, education, occupation, and poverty, as measured by family possession of Below Poverty Line (BPL) card, were used to look at the effect of intervention on hospitalization and mortality. While the CCP intervention had no effect on 21-day mortality (OR 0.64; 95% CI, 0.19 to 2.12), it was associated with a 48% reduction in 21-day hospitalization (OR 0.52; 95% CI, 0.31 to 0.90). Conclusion: COVID-19 CCP teletraining intervention reduced the rate of hospitalization, potentially reducing the burden on hospitals.

Alzheimer's Disease: Treatment of Multi-Factorial Disorders with Multi- Target Approach.

Alzheimer's Disease (AD) is a common neurodegenerative disorder that is almost incurable with the existing therapeutic interventions. Due to the high-risk factors associated with this disease, there is a global pursuit of new anti-AD agents. Herein, we explore the biochemical pathways which are responsible for the initiation/propagation of the disease. It is observed that out of the two isoforms of ß-secretase, ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) and ß-site amyloid precursor protein cleaving enzyme 2 (BACE2) present in the brain, BACE1 plays the predominant role in the commencement of AD. Moreover, the catalytic activities of acetylcholinesterase and butyrylcholinesterase regulate the concentration of neurotransmitters, and they are needed to be kept under control during the signs of AD. Hence, these two enzymes also serve as potential targets for the treatment of AD patients. Keeping in view the multifactorial nature of the disease, we also reviewed the multitarget approach for the treatment of AD. It is tried to identify the common structural features of those molecules which act on different cellular targets during AD therapy.

Proline based rationally designed peptide esters against dipeptidyl peptidase-4: Highly potent anti-diabetic agents.

With the target to develop small molecules based anti-diabetic agents, we, herein, report the design, synthesis and biological studies on Lys-Pro and Gly-Pro esters, and a Phe-Pro-Phe tripeptide inhibiting the activity of glycoprotein dipeptidyl peptidase-4 (DPP-4). Since DPP-4 cleaves the glucagon like peptide (GLP-1) and glucose dependent insulinotropic polypeptide (GIP) hormones which are responsible for inducing insulin secretion, the results of present studies could be significant in making control over glycemia. The structural analysis of DPP-4 and its binding mode with the substrate as well as the reported inhibitors provided the background for the design of new molecules. Among the 17 compounds screened against DPP-4, 14 compounds displayed IC50 better than the known drug Sitagliptin. Collectively, a highly encouraging set of molecules was identified that may prove as the clinical candidates for the treatment of diabetes.

Garg scoring system to predict long-term healing in cryptoglandular anal fistulas: a prospective validation study.

Purpose: Complex anal fistulas can recur after clinical healing, even after a long interval which leads to significant anxiety. Also, ascertaining the efficacy of any new treatment procedure becomes difficult and takes several years. We prospectively analyzed the validity of Garg scoring system (GSS) to predict long-term fistula healing. Methods: In patients operated for cryptoglandular anal fistulas, preoperative magnetic resonance imaging (MRI) and postoperative MRI was done at 3 months to assess fistula healing. Scores as per the GSS were calculated for each patient at 3 months postoperatively and correlated with long-term healing to check the accuracy of the scoring system. Results: Fifty-seven patients were enrolled, but 50 were finally included (7 were excluded). These 50 patients (age, 41.2± 12.4 years; 46 males) were followed up for 12 to 20 months (median, 17 months). Forty-seven patients (94.0%) had complex fistulas, 28 (56.0%) had recurrent fistulas, 48 (96.0%) had multiple tracts, 20 (40.0%) had horseshoe tracts, 15 (30.0%) had associated abscesses, 5 (10.0%) were suprasphincteric, and 8 (16.0%) were supralevator fistulas. The GSS could accurately predict long-term healing (specificity and high positive predictive value, 31 of 31 [100%]) but was not very accurate in predicting non-healing (negative predictive value, 15 of 19 [78.9%]). The sensitivity in predicting healing was 31 of 35 (88.6%). Conclusion: GSS accurately predicts long-term fistula with a high positive predictive value (100%) but is less accurate in predicting non-healing. This scoring system can help allay anxiety in patients and facilitate the early validation of innovative procedures for anal fistulas.

Non-Locatable Internal Opening in Anal Fistula Associated with Acute Abscess and Its Definitive Management by Garg Protocol.

Background: Definitive management of acute fistula-abscess (anal fistulas associated with acute abscess) is gaining popularity against the two-staged approach (early abscess drainage with deferred fistula management). However, locating an internal opening (IO) in acute fistula-abscess can be difficult. A recent protocol (Garg protocol) has been shown to be effective in managing anal fistulas with non-locatable IO. Purpose: To test the efficacy of the Garg protocol in managing acute fistula-abscess with non-locatable IO. Methods: Patients with acute fistula-abscess operated by a definitive procedure were included. A preoperative MRI was done in all patients. Patients in whom the IO was non-locatable after clinical, MRI, and intraoperative examination were managed by the three-step Garg protocol. Garg protocol: 1) Reassessment of MRI; 2) In non-horseshoe fistulas, the IO was assumed to be at the point where the fistula tract reached closest to the sphincter-complex; 3) In horseshoe fistulas, the IO was assumed to be located in the midline (anterior or posterior as per the horseshoe location). Low fistulas were treated by fistulotomy and high fistulas by a sphincter-sparing procedure. The long-term healing rate and change in continence (Vaizey scores) were evaluated. Results: A total of 201 patients with acute fistula-abscess were operated over six years, and 19 were lost to follow-up. A total of 182 patients (154-males) were followed up (median-37 months). The IO was locatable in 133/182 (73.1%) (control group) and was non-locatable in 49/182 (26.9%) (study group). The study group was managed as per the Garg protocol. The age, sex-ratio, and fistula parameters were comparable in both groups. The long-term healing rate was 112/133(84.2%) in the IO-locatable group and 43/49 (87.8%) in the IO-non-locatable group (p=0.64, not-significant). The objective continence scores did not change significantly after surgery in both groups. Conclusion: Acute fistula-abscess with non-locatable IO can be managed successfully by the Garg protocol without any risk of incontinence.

Efficacy of Kegel exercises in preventing incontinence after partial division of internal anal sphincter during anal fistula surgery.

BACKGROUND: The transanal opening of intersphincteric space (TROPIS) procedure, performed to treat complex anal fistulas, preserves the external anal sphincter (EAS) but involves partial incision of the internal anal sphincter (IAS). AIM: To ascertain the incidence of incontinence after the division of the IAS as is done in TROPIS and to evaluate whether regular Kegel exercises (KE) in the postoperative period can prevent incontinence due to IAS division. METHODS: Patients operated on for high complex fistulas and having no preoperative continence problem (score = 0) were included in the study. All patients were operated on by the TROPIS procedure and were recommended KE (pelvic contraction exercises) 50 times/day. KE were commenced on the 10th postoperative day and continued for 1 year. Incontinence was evaluated objectively (by modified Vaizey's scores) in the immediate postoperative period (Pre-KE group) and on long-term follow-up (Post-KE group). The incontinence scores in both groups were compared to evaluate the efficacy of KE. RESULTS: Of 102 anal fistula patients operated on between July 2018 and July 2020 were included in this study. There were 90 males, the mean age was 42.3 ± 12.8, and the median follow-up was 30 mo (18-42 mo). Three patients were lost to follow-up. There were 65 recurrent fistulas, 92 had multiple tracts, 42 had associated abscess, 46 had horseshoe fistula and 34 were supralevator fistulas. All were magnetic resonance imaging-documented high fistulas (> 1/3 EAS involved). Overall incontinence occurred in 31% patients (Pre-KE group) with urge and gas incontinence accounting for the majority of cases (28.3%). The mean incontinence scores in the Pre-KE group were 1.19 ± 1.96 (in 31 patients, solid = 0, liquid = 7, gas = 8, urge = 24) and in the Post-KE group were 0.26 ± 0.77 (in 13 patients, solid = 0, liquid = 2, gas = 3, urge = 10) (P = 0.00001, t-test). CONCLUSION: Division of the IAS led to incontinence, mainly urge incontinence, and also to a mild degree of gas and liquid incontinence. However, regular KE led to a significant reduction in incontinence (both in the number of affected patients and the severity of scores in these patients).

TUXEDO: A phase I/II trial of cetuximab with chemoradiotherapy in muscle-invasive bladder cancer.

OBJECTIVE: To assess feasibility and preliminary efficacy of adding cetuximab to standard chemoradiotherapy for muscle-invasive bladder cancer. PATIENTS AND METHODS: TUXEDO was a prospective, single-arm, open-label, phase I/II trial conducted in six UK hospitals. Cetuximab was administered with an initial loading dose of 400mg/m2 on day 1 of week -1, and then 7-weekly doses of 250mg/m2 . Radiotherapy schedule was 64Gy/32F with day 1 mitomycin C (12g/m2 ) and 5-fluorouracil (500mg/m2 /day) over days 1-5 and 22-26. Patients with T2-4aN0M0 urothelial cancer and a performance status (PS) of 0-1 were eligible. Prior neoadjuvant therapy was permitted. The phase I primary outcome was impact on radiotherapy treatment completion and toxicity experienced during treatment. The phase II primary outcome was local control at three-months post-treatment. ISRCTN identifier: 80733590. RESULTS: Between Sept-2012 and Oct-2016, 33 patients were recruited; 7 in phase I, 26 in phase II. Three patients in phase II were subsequently deemed ineligible and received no trial therapy. Eight patients discontinued cetuximab due to adverse effects. Median age of patients was 70.1 years (range 60.6-75.1), 20 were PS 0, 27 male and 26 had already received neoadjuvant chemotherapy. In phase I, all patients completed planned radiotherapy, with no delays or dose reductions. Of the 30 evaluable patients in phase II, 25 had confirmed local control 3-months post treatment (77%, 95% CI: 58-90). During the trial there were 18 serious adverse events. The study was halted due to slow accrual. CONCLUSION: Phase I data demonstrate it is feasible and safe to add cetuximab to chemoradiotherapy. Exploratory analysis of phase II data provides evidence to consider further clinical evaluation of cetuximab in this setting.

Including video and novel parameter-height of penetration of external anal sphincter-in magnetic resonance imaging reporting of anal fistula.

The main purpose of a radiologist's expertise in evaluation of anal fistula magnetic resonance imaging (MRI) is to benefit patients by decreasing the incontinence rate and increasing the healing rate. Any loss of vital information during the transfer of this data from the radiologist to the operating surgeon is unwarranted and is best prevented. In this regard, two methods are suggested. First, a short video to be attached with the standardized written report highlighting the vital parameters of the fistula. This would ensure minimum loss of information when it is conveyed from the radiologist to the operating surgeon. Second, inclusion of a new parameter, the amount of external sphincter involvement by the anal fistula. This parameter is usually not included in the MRI report. This can be evaluated as the height of penetration of the external anal sphincter (HOPE) by the fistula. The external anal sphincter plays a pivotal role in maintaining continence. This parameter (HOPE) is distinct from the 'height of internal opening' and assumes immense importance as its knowledge is paramount to prevent damage to the external anal sphincter by the surgeon during surgery.

Lignocellulolytic enzymes from Aspergillus allahabadii for efficient bioconversion of rice straw into fermentable sugars and biogas.

The study was aimed at developing lignocellulolytic strain capable of efficient hydrolysis of mild alkali deacetylated (MAD) rice straw. The valorisation of lignin rich black liquor obtained during pre-treatment of rice straw into biogas was also evaluated. Study reports highly proficient cellulolytic Aspergillus allahabadii strain harbouring a spectrum of CAZymes based on comparative genome wide analysis that was subjected to strain breeding for developing a hyper producing strain. The secretome analysis showed up-modulation and several folds increase in the CAZyme activities in the culture extracts of the developed strain MAN 40 when compared to parent. The cellulolytic cocktail of the developed strain showed 1.52 folds higher saccharification of MAD rice straw when compared to Cellic CTec 3. Moreover, in-situ addition of cellulases derived from developed strains resulted in ∼3.7 folds higher methane production during anaerobic digestion of mixture of lignin rich black liquor and differently treated rice straw.

Guidelines to diagnose and treat peri-levator high-5 anal fistulas: Supralevator, suprasphincteric, extrasphincteric, high outersphincteric, and high intrarectal fistulas.

Supralevator, suprasphincteric, extrasphincteric, and high intrarectal fistulas (high fistulas in muscle layers of the rectal wall) are well-known high anal fistulas which are considered the most complex and extremely challenging fistulas to manage. Magnetic resonance imaging has brought more clarity to the pathophysiology of these fistulas. Along with these fistulas, a new type of complex fistula in high outersphincteric space, a fistula at the roof of ischiorectal fossa inside the levator ani muscle (RIFIL), has been described. The diagnosis, management, and prognosis of RIFIL fistulas is reported to be even worse than supralevator and suprasphincteric fistulas. There is a lot of confusion regarding the anatomy, diagnosis, and management of these five types of fistulas. The main reason for this is the paucity of literature about these fistulas. The common feature of all these fistulas is their complete involvement of the external anal sphincter. Therefore, fistulotomy, the simplest and most commonly performed procedure, is practically ruled out in these fistulas and a sphincter-saving procedure needs to be performed. Recent advances have provided new insights into the anatomy, radiological modalities, diagnosis, and management of these five types of high fistulas. These have been discussed and guidelines formulated for the diagnosis and treatment of these fistulas for the first time in this paper.

Epoxides: Developability as active pharmaceutical ingredients and biochemical probes.

Epoxide functional group is a part of several natural as well as synthetic compounds. Irrespective of the role of epoxide group in drug efficacy and the use of epoxide compounds as tool molecules, uncertainty prevails over concerns associated with the reactivity of this functional group. Herein, we compile information about epoxide-based medicinal compounds and biochemical probes and look into the related advantages and challenges of the epoxide functional group. As a whole, this study is focussed on analyzing the strategies which have been adopted for the successful development of epoxide-based compounds within drug discovery programs.

Comparison of different methods to manage supralevator rectal opening in anal fistulas: A retrospective cohort study.

INTRODUCTION: Supralevator fistula-in-ano are difficult to manage. If these fistulas have an additional supralevator internal-opening in rectum apart from the primary internal-opening at the dentate line, then the management becomes even more difficult. There is no literature/guidelines available on the management of supralevator rectal opening (SRO). METHODS: All consecutive supralevator fistula-in-ano patients having a SRO were retrospectively analyzed. The operative management of SRO in these fistulas was reviewed. All the fistulas were managed by the same procedure, transanal opening of intersphincteric space (TROPIS). The latter was a modification of LIFT (ligation of intersphincteric tract) procedure in which the intersphincteric tract was opened-up in the rectum rather than ligated (as is done in LIFT). The SRO was managed in three ways, group-1:SRO was laid-open into the rectum in continuity with the primary opening at dentate line, group-2:the mucosa around SRO was cauterized, group-3:nothing could be done to SRO. RESULTS: Out of 836 patients operated between 2015 and 2020, 138 patients (16.5%) had supralevator extension. Amongst these, 23/138 (16.6%) patients had a SRO. 2 patients were excluded (short follow-up) and 21 patients were included in the analysis. 12/13(92%) patients in group-1, 4/5 (80%) patients in group-2 and 2/3(67%) patients in group-3 got healed (p=0.47, Chi-square test). The overall healing rate was 18/21(86%). CONCLUSIONS: The supralevator rectal opening (SRO) heals well irrespective of the method utilized. Thus, proper management of the primary opening at the dentate line holds the key to fistula healing and SRO is perhaps not much responsible for persistence of the fistula. However, more studies are needed to corroborate these findings.