Development of whey protein beverage incorporating encapsulated probiotic strain Lactiplantibacillus rhamnosus NCDC 347 and its physico-chemical characteristics.

In the present study, encapsulated strain Lactiplantibacillus rhamnosus NCDC 347 was used to prepare a novel whey protein-based beverage. The encapsulation process utilized skimmed milk powder matrix and evaluated strain viability, physico-chemical properties, sensory assessment, and shelf-life stability. Encapsulated L. rhamnosus NCDC 347 within skim milk powder maintained viability at 8.0 log CFU/g, forming spherical microcapsules with 1-12 µm concavities. Probiotic addition to whey protein beverages maintained pH and acidity within desired ranges. Physico-chemical analysis showed protein content of 8.71 ± 0.21 % to 10.05 ± 0.42 %, fat content of 0.56 ± 0.24 % to 0.67 ± 0.13 %, viscosity of 5.14 pa/s, and total soluble solids (TSS) of 14.42 ± 0.31 to 16.16 ± 0.23° Brix. The shelf-life study revealed that the beverage remained stable for up to 90 days with no significant changes (p > 0.05) in sensory analysis. The sensory analysis scored the test sample's acceptability at 7.3 ± 0.41. The protein-rich probiotic drink exhibited favorable sensory qualities. Overall, incorporating encapsulated probiotic strain L. rhamnosus NCDC 347 into whey protein beverages could address daily protein requirements and enhance health.

Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers.

The introduction of spacers in coating steroid protein complexes and/or enzyme conjugates or immunogens is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We investigated the impact of different homobifunctional spacers, ranging in atomic length from 3 to 10, on the sensitivity and specificity of prednisolone (PSL) enzyme immunoassays. In this study, four homo-bifunctional spacers, namely, carbohydrazide (CH), adipic acid dihydrazide (ADH), ethylene diamine (EDA), and urea (U), were incorporated between PSL and horseradish peroxidase (HRP) for preparing the enzyme conjugate with an aim to improve the sensitivity of the assay without compromising assay specificity. The assays were developed using these enzymes conjugated with antibodies raised against the PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of a bridge in the enzyme conjugate was 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL, and 0.018 ng/mL with ADH, CH, EDA, and urea as a spacer, respectively. Among the four combinations, the PSL-21-HS-BSA-antibody with PSL-21-HS-U-HRP-enzyme conjugate gave better sensitivity and less cross-reaction. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32%-102.50%. The intra and inter-assay CV% was< 8.46%. The PSL concentration was estimated in the serum samples of patients on PSL treatment. The serum PSL values obtained by this method correlated well with the commercially available kit (r2 = 0.98). The present study suggests that the nature of the spacer is related to assay sensitivity and not the spacer length.

Rheumatology and palliative care: needs and opportunities.

Palliative care (PC) has expanded to medical conditions beyond its conventional scope of terminal malignancy and end-stage organ failure. This editorial showed our opinion in care model for the integration of PC into rheumatology and the growing needs of both rheumatology and PC services in view of increasing comorbidities and novel therapies. We anticipate an escalating demand for PC in this special group of patients who have concomitant long-standing systemic rheumatic diseases and age-related comorbidities. In addition, patients with advanced malignancy who develop rheumatological problems and require PC is also an emerging area of service need.

COVID-19db linkage maps of cell surface proteins and transcription factors in immune cells

The highly contagious SARS-CoV-2 and its associated disease (COVID-19) are a threat to global public health and economies. To develop effective treatments for COVID-19, we must understand the host cell types, cell states and regulators associated with infection and pathogenesis such as dysregulated transcription factors (TFs) and surface proteins, including signaling receptors. To link cell surface proteins with TFs, we recently developed SPaRTAN (Single-cell Proteomic and RNA-based Transcription factor Activity Network) by integrating parallel single-cell proteomic and transcriptomic data based on Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), which contains gene cis-regulatory information. We apply SPaRTAN to CITE-seq datasets from patients with varying degrees of COVID-19 severity and healthy controls to identify the associations between surface proteins and TFs in host immune cells. Here, we present COVID-19db of Immune Cell States (https://covid19db.streamlit.app/), a web server containing cell surface protein expression, SPaRTAN-inferred TF activities, and their associations with major host immune cell types. The data include four high-quality COVID-19 CITE-seq datasets with a toolset for user-friendly data analysis and visualization. We provide interactive surface protein and TF visualizations across major immune cell types for each dataset, allowing comparison between various patient severity groups for the discovery of potential therapeutic targets and diagnostic biomarkers.

Use of US Food and Drug Administration Expedited Drug Development and Review Programs by Orphan and Nonorphan Novel Drugs Approved From 2008 to 2021.

Importance: The US Food and Drug Administration (FDA) has 4 programs that can be used alone or in combination to expedite drug availability: Accelerated Approval, Breakthrough Therapy, Fast Track, and Priority Review. Drugs using these programs can include novel drugs, which do not contain a previously FDA-approved active moiety, and orphan drugs, intended for diseases or conditions affecting fewer than 200 000 people; to date, no comprehensive evaluation of how these programs have been used in combination has been published. Objective: To assess how often and in what combinations expedited programs are used in the development and review of approved novel biologics and small-molecule drugs, stratified by orphan drug status and indication. Design, Setting, and Participants: This cross-sectional study evaluated all novel drugs that were FDA approved between January 1, 2008, and December 31, 2021. Main Outcomes and Measures: The main outcome was the frequency with which expedited programs were used and in what combinations, stratified by orphan drug status and drug type (small molecule vs therapeutic biologic). The unit of analysis was the novel drug-indication pair because a drug can be approved for multiple indications, each of which may use a different expedited program or differ in orphan drug status. Results: The study included 581 novel drug-indication pairs approved during the 14-year study period; 252 (43.4%) were orphan drugs, 139 (23.9%) were therapeutic biologics, and 442 (76.1%) were small-molecule drugs. Use of at least 1 expedited program increased from 11 of 26 drug-indication pairs (42.3%) in 2008 to 41 of 55 (74.5%) in 2021. Of the 363 approved drug-indication pairs using at least 1 expedited program, 225 (62.0%) were orphan drugs; at least 1 expedited program was used by 97 of the 139 approved biologic drugs (69.8%) and by 266 of the 442 approved small-molecule drugs (60.2%). Eighty-two of the 581 novel drug-indication pairs (14.1%) used the Accelerated Approval Program; of those, 65 (79.3%) were oncology drugs and 70 (85.4%) had an orphan designation. Conclusions and Relevance: The study showed that use of the FDA's expedited programs to bring novel drugs to market in the US increased from 2008 to 2021. The findings suggest that this trend is likely to continue.

Germination induced changes in bioactive compounds and nutritional components of millets.

The present study was conducted to compare the nutritional quality and the concentration of bioactive compounds in the flours from dehusked and germinated foxtail, barnyard, and little millets. Germinated millet flours showed significantly higher protein content (11.79-33.49%), total free amino acid content (66-334.87%) and protein solubility (13.83-34%), compared to the dehusked millet flours. The total phenols and flavonoids in the flours from the three germinated millets were significantly higher by about 142.36 and 437.20%, respectively, compared to the flours from the dehusked millets. Mineral content was also found to be higher in the flours from germinated millets in comparison to the flours from dehusked millets. The results of this study showed that the flours from the germinated millets have the potential for their application in development of novel products,because of their enhanced nutritional value. The extracts from the germinated millets have the potential for use as functional ingredients in the development of novel processed fruit beverages.

Hydrolysis of peanut (Arachis hypogea L) protein concentrate by fungal crude protease extract: effect on structural, functional and in-vitro protein digestibility.

Peanut protein concentrates (PPCs) were subjected to hydrolysis by crude protease extract (CPE) obtained from three fungi viz; Rhizopus oligosporus, Trichoderma reesei, and Aspergillus oryzae and the effect on structural, functional and in-vitro protein digestibility (IVPD) properties were studied. Particle size was found significantly (p ≤ 0.05) lower in hydrolyzed samples than un-treated samples. Fourier transform infrared spectroscopy (FTIR) spectrum of hydrolyzed samples displayed intense absorbance peaks in the wavelength ranging from 1500 to 2600 cm-1. Peanut protein concentrates hydrolyzed by CPE from R. oligosporus showed higher surface hydrophobicity (564.18). Total sulfhydryl content was found lower in all the hydrolyzed samples whereas, reverse trend was observed for exposed sulfhydryl content. The structural changes simultaneously affected the functional and IVPD attributes of hydrolyzed PPCs. In comparison to the PPCs hydrolysed using crude extracts from T. reesei and R. oligosporus, PPCs hydrolysed by A, oryzae showed higher solubility, water and oil binding capacity, foaming capacity and foam stability. Higher IVPD values of 86.70% was also found in PPCs hydrolyzed with CPE of A. oryzae. The study established that CPE hydrolysis of PPCs has potential for scale-up studies and may serve as a cost effective alternative to protein hydrolysis with pure enzymes.

Immune and Clotting Dysfunction Detected in Saliva and Blood Plasma after COVID-19.

A growing number of studies indicate that coronavirus disease 2019 (COVID-19) is associated with inflammatory sequelae, but molecular signatures governing the normal vs. pathologic convalescence process have not been well-delineated. We characterized global immune and proteome responses in matched plasma and saliva samples obtained from COVID-19 patients collected between 4-6 weeks after initial clinical symptoms resolved. Convalescent subjects showed robust IgA and IgG responses and positive antibody correlations between matched saliva and plasma samples. However, global shotgun proteomics revealed persistent inflammatory patterns in convalescent samples including dysfunction of salivary innate immune cells and clotting factors in plasma (e.g., fibrinogen and antithrombin), with positive correlations to acute COVID-19 disease severity. Saliva samples were characterized by higher concentrations of IgA, and proteomics showed altered pathways that correlated positively with IgA levels. Our study positions saliva as a viable fluid to monitor immunity beyond plasma to document COVID-19 immune, inflammatory, and coagulation-related sequelae.

Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, predictors of outcomes within severe COVID-19 remain to be comprehensively elucidated. Recently, we identified divergent monocyte states as predictors of outcomes within severe COVID-19, but corresponding humoral profiles of risk have not been delineated. Furthermore, the nature of antibodies (Abs) directed against viral antigens beyond the spike protein or endemic coronavirus antigens and their associations with disease severity and outcomes remain poorly defined. We performed deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients admitted to the ICU. The profiles consisted of canonical (S, RBD, N) and non-canonical (orf3a, orf8, nsp3, nps13 and M) antigenic specificities. Notably, multivariate machine learning (ML) models, generated using profiles of Abs directed against canonical or non-canonical antigens, were equally discriminative of recovery and mortality COVID-19 outcomes. In both ML models, survivors were associated with increased virus-specific IgA and IgG3 antibodies and with higher antigen-specific antibody galactosylation. Intriguingly, pre-pandemic healthy controls had cross-reactive Abs directed against nsp13 which is a conserved protein in other alpha and beta coronaviruses. Notably, higher levels of nsp13-specific IgA antibodies were associated with recovery in severe COVID-19. In keeping with these findings, a model built on Ab profiles for endemic coronavirus antigens was also predictive of COVID-19 outcome bifurcation, with higher levels of IgA and IgG3 antibodies against OC43 S and NL63 S being associated with survival. Our results suggest the importance of Abs targeting non-canonical SARS-CoV-2 antigens as well as those directed against endemic coronaviruses in favorable outcomes of severe COVID-19.

Preoperative COVID-19 Testing for Elective Ophthalmological Procedure in a Tertiary Health Care Centre: Our Experience During the Pandemic.

BACKGROUND: To study the prevalence of coronavirus infection among asymptomatic patients requiring eye surgery and the role of screening in prevention of spread of infection among the healthcare workers. DESIGN: The prospective observational study was conducted in the Ophthalmology department of a tertiary care center in Delhi from September 2020 to December 2020. SETTING: Patients requiring elective ophthalmological procedures in a tertiary care hospital were screened for coronavirus using the RT-PCR method. Testing methods and results were documented. RESULTS: Among the 218 asymptomatic patients posted for elective surgery in that period, 16 (7.3%) were found to be positive for COVID-19. Those who tested positive were advised home isolation and surgery was postponed for the next 14 days. No complications were reported in these patients. One health-care worker also tested positive for COVID-19 during our study period. CONCLUSION: In our study, 1 out of 14 asymptomatic patients were found to be carriers for the novel virus. Asymptomatic COVID-infected patients may lead to transmission of the virus inside the hospital among the visiting patients and hospital staff while they have no adverse effect on the surgery and its outcome.

Optic perineuritis.

Optic perineuritis (OPN) is a rare inflammatory disorder in which the inflammation is confined to optic nerve sheath. It can be idiopathic or secondary to underlying systemic autoimmune disorder. It usually presents with unilateral progressive diminution of vision with pain on eye movements and optic disc oedema. Hence, clinically OPN mimics optic neuritis resulting in delayed diagnosis and suboptimal treatment. In contrast to optic neuritis, patients with OPN are usually of older age group and more likely show sparing of central vision. MRI is an important tool for diagnosis of OPN apart from optic nerve sheath biopsy. Perineural enhancement on MRI is diagnostic of OPN. Oral corticosteroid therapy gives dramatic and rapid improvement in signs and symptoms. Rapid tapering of steroids increases the risk of relapse. Overall, prognosis of OPN is generally good if adequate treatment is given timely.

Fungal Genomic Resources for Strain Identification and Diversity Analysis of 1900 Fungal Species.

Identification and diversity analysis of fungi is greatly challenging. Though internal transcribed spacer (ITS), region-based DNA fingerprinting works as a "gold standard" for most of the fungal species group, it cannot differentiate between all the groups and cryptic species. Therefore, it is of paramount importance to find an alternative approach for strain differentiation. Availability of whole genome sequence data of nearly 2000 fungal species are a promising solution to such requirement. We present whole genome sequence-based world's largest microsatellite database, FungSatDB having >19M loci obtained from >1900 fungal species/strains using >4000 assemblies across globe. Genotyping efficacy of FungSatDB has been evaluated by both in-silico and in-vitro PCR. By in silico PCR, 66 strains of 8 countries representing four continents were successfully differentiated. Genotyping efficacy was also evaluated by in vitro PCR in four fungal species. This approach overcomes limitation of ITS in species, strain signature, and diversity analysis. It can accelerate fungal genomic research endeavors in agriculture, industrial, and environmental management.

Contents of US Food and Drug Administration Refuse-to-File Letters for New Drug Applications and Efficacy Supplements and Their Public Disclosure by Applicants.

Importance: Before reviewing drug applications, the US Food and Drug Administration (FDA) conducts "filing reviews" to assess whether they are complete enough for full review. If the applications are incomplete, the FDA issues refuse-to-file (RTF) letters identifying deficiencies. The FDA does not make these RTF letters public at the time of issuance. Why the FDA issues RTF letters and how often the letters and their contents are made publicly available are unknown. Objectives: To quantitatively analyze the FDA's reasons for issuing RTF letters and assess the public transparency of RTF letters and their contents. Design and Setting: This cross-sectional study analyzes RTF letters issued in response to new drug applications and efficacy supplements (applications for new indications or patient populations for already approved drugs) submitted to the FDA between January 1, 2008, and December 31, 2017. Statistical analysis was conducted in July 2019. Main Outcomes and Measures: Two types of information were extracted and cataloged from RTF letters: (1) the reasons why the FDA refused to file applications and (2) the FDA comments that, while not a basis for RTF letters, conveyed important information to applicants. The extent to which applicants publicly disclosed the FDA's refusal reasons were also assessed. Results: The study included 103 RTF letters containing a total of 644 identified FDA refusal reasons. Among the 2475 applications that the FDA received during the study time frame, 98 (4.0%) received RTF letters. Overall, 84.5% (544 of 644) of the refusal reasons were for scientific deficiencies; most reasons were related to drug efficacy and safety (196 [30.4%]) and drug quality (125 [19.4%]). The remaining 15.5% of refusal reasons (100 of 644) were for application organization deficiencies or legal issues. A total of 26.2% of the RTF letters (27 of 103) identified presubmission advice from the FDA that applicants did not follow; the most frequently ignored advice was related to clinical trial design (33.3% [9 of 27]), followed by product chemistry and manufacturing (25.9% [7 of 27]). Applicants publicly disclosed the existence of 16 of 103 RTF letters (15.5%); however, only 5.4% of applicant-disclosed reasons (35 of 644) matched the refusal reasons that the FDA had provided in the RTF letters. Conclusions and Relevance: This cross-sectional study found that the FDA refused to file applications for substantive reasons related to quality, safety, and efficacy, and applicants' disclosure of those reasons was incomplete. This work sheds light on the FDA's regulatory decision-making processes and the RTF reasons that could delay availability of therapies to patients.

Bifurcated monocyte states are predictive of mortality in severe COVID-19

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection presents with varied clinical manifestations1, ranging from mild symptoms to acute respiratory distress syndrome (ARDS) with high mortality2,3. Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in severe COVID-19. We performed high-dimensional cellular and molecular profiling of blood and respiratory samples from critically ill COVID-19 patients to define immune cell genomic states that are predictive of outcome in severe COVID-19 disease. Critically ill patients admitted to the intensive care unit (ICU) manifested increased frequencies of inflammatory monocytes and plasmablasts that were also associated with ARDS not due to COVID-19. Single-cell RNAseq (scRNAseq)-based deconvolution of genomic states of peripheral immune cells revealed distinct gene modules that were associated with COVID-19 outcome. Notably, monocytes exhibited bifurcated genomic states, with expression of a cytokine gene module exemplified by CCL4 (MIP-1{beta}) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1{beta} and CXCL10 levels in plasma, respectively. Monocytes expressing genes reflective of these divergent modules were also detectable in endotracheal aspirates. Machine learning algorithms identified the distinctive monocyte modules as part of a multivariate peripheral immune system state that was predictive of COVID-19 mortality. Follow-up analysis of the monocyte modules on ICU day 5 was consistent with bifurcated states that correlated with distinct inflammatory cytokines. Our data suggests a pivotal role for monocytes and their specific inflammatory genomic states in contributing to mortality in life-threatening COVID-19 disease and may facilitate discovery of new diagnostics and therapeutics.

Topical vs Peribulbar Anesthesia: Comparison of Anterior Chamber Depth and the Resultant Visual Outcome After Phacoemulsification.

PURPOSE: To compare anterior chamber depth and postoperative visual outcome in patients undergoing phacoemulsification under topical vs peribulbar anesthesia. MATERIALS AND METHODS: Prospective, randomized, comparative observational study.Sample size: 110 eyes with 55 eyes each. Group I: Patients undergoing phacoemulsification under topical anesthesia.Group II: Patients undergoing phacoemulsification under peribulbar anesthesia.Once patients were selected, baseline a standard ophthalmic examination was done including best-corrected visual acuity (BCVA), refraction, IOP by NCT, anterior segment evaluation with slit lamp biomicroscopy, keratometry, axial length, and ACD measurement by IOL master. Post-operatively, the patients were reviewed at day 1, week 1, week 6 for the same parameters.Statistics:Quantitative variables: Paired and unpaired t-test.Qualitative variables: Chi square test.A P-value of <0.05 was considered statistically significant. RESULTS: Inter-group comparisons of post-operative change in ACD at 1-week post-op (P-value<0.001) and 6-week post-op (P-value<0.001) were statistically significant when compared to the pre-op values. The mean spherical equivalent in group I was 0.27±0.26 d and that in group II was 1±0.32 d at 1-week post-op. The mean spherical equivalent in group I was 0.23±0.20 d, while that in group II was 0.85±0.64 d at 6-week post-op. This difference was statistically significant both at 1-week post-op (P-value=0.002) and 6-week post-op (P-value<0.001). CONCLUSION: Post-phacoemulsification, the ACD is more after peribulbar anesthesia as compared to topical anesthesia. Post-op refractive outcome is better with the use of topical anesthesia.

Forceps-assisted, 10-0 monofilament nylon, suture-guided capsular tension ring insertion in subluxated cataracts.

Capsular tension ring (CTR) and iris hooks have proved to be useful devices in cataract surgery in cases of zonular weakness and dialysis. We would like to describe the use of monofilament 10-0 nylon (MFN)- guided CTR insertion in a subluxated cataractous lens. Here, the MFN is passed through the distal trailing eyelet of the CTR (simple or Morcher's depending on the size of the defect and subluxation) for controlled insertion of the distal eyelet of the CTR beneath the anterior capsulorhexis margin and safe retrieval of CTR in case of an iatrogenic posterior capsular tear during CTR insertion or phacoemulsification.