A comprehensive study on the identification and characterization of degradation products of lipoglycopeptide Dalbavancin using LC and LC-HRMS/MS.

Dalbavancin is the second-generation approved semisynthetic lipoglycopeptide by the United States Food and Drug Administration (USFDA) for the treatment of acute bacterial skin and skin-structure infections. Unlike other lipoglycopeptides, the stability behavior of Dalbavancin was least explored, which is a prerequisite. The current study endeavors to elucidate the oxidative and hydrolytic stability behavior of Dalbavancin by exposing the drug to oxidative, acidic, and basic stress conditions. A simple liquid chromatography (LC) method was developed, where significant resolution between Dalbavancin, its homologs, and the generated degradation products was achieved. Seven degradation products were identified under acidic, basic, and oxidative stress conditions. Using liquid chromatography and high-resolution mass spectrometry (LC-HRMS), MS/MS studies, the generated degradation products were identified and characterized. Formation of isomeric degradation products was identified especially upon exposure to basic stress conditions. The mechanistic fragmentation pathway for the seven degradation products was established, and the chemical structure for the identified degradation products was elucidated. The results strongly suggest that Dalbavancin is highly susceptible to degradation under oxidative and hydrolytic stress conditions. This study provides insights into the hydrolytic and oxidative stability of Dalbavancin, which can be employed during drug development and discovery in synthesizing relatively stable analogs.

Biochemical characterization of a novel acid-active endopolygalacturonase for pectin depolymerization, pectic-oligomer production, and fruit juice clarification.

Endopolygalacturonases are crucial pectinases known for their efficient and sustainable pectin depolymerization activities. The present study identified a novel gene encoding endopolygalacturonase from an acidic mine tailing metagenome. The putative gene showed a maximum identity of 67.55 % with an uncharacterized peptide sequence from Flavobacterium fluvii. The gene was cloned and expressed in a heterologous host, E. coli. Biochemical characterization of the novel endopolygalacturonase enzyme variant (EPHM) showed maximum activity at 60 °C and at 5.0 pH, while retaining 50 % activity under the temperature and pH range of 20 °C to 70 °C for 6 h, and 3.0 to 10.0 for 3 h, respectively. The enzyme exhibited tolerance to different metal ions. EPHM was characterized for the depolymerization of methylated pectin into pectic oligosaccharides. Further, its utility was established for fruit juice clarification, as endorsed by high transmittance, significant viscosity reduction, and release of reducing sugars in the treated fruit juice samples.

Enantiomeric purity of synthetic therapeutic peptides: A review.

Synthetic therapeutic peptides are a complex and popular class of pharmaceuticals. In recent years, peptides with proven therapeutic activity have gained significant interest in the market. The determination of synthetic peptide enantiomeric purity plays a critical role in the evaluation of the quality of the medicine. Since racemization is one of the most common side reactions occurring in AAs or peptides, enantiomeric impurities such as D-isomers can form during the peptide synthesis or can be introduced from the starting materials (e.g., AAs). The therapeutic effect of a synthetic or semi-synthetic bioactive peptide molecule depends on its AA enantiomeric purity and secondary/tertiary structure. Therefore, the enantiomeric purity determination for synthetic peptides is supportive for interpreting unwanted therapeutic effects and determining the quality of synthetic peptide therapeutics. However, enantiomeric purity analysis encounters formidable analytical challenges during chromatographic separation, as D/L isomers have identical physical-chemical properties except stereochemical configuration. To ensure peptides AA stereochemical configuration whether in the free or bound state, sensitive and reproducible quantitative analytical method is mandatory. In this regard, numerous analytical techniques were emerged for the quantification of D-isomeric impurities in synthetic peptides, but still, very few reports are available in the literature. Thus, the purpose of this paper is to provide an overview of the importance, regulatory requirements, and various analytical methods used for peptide enantiomeric purity determination. In addition, we discussed the available literature in terms of enantiomeric impurity detection, common hydrolysis procedural aspects, and different analytical strategies used for sample preparation.

Development of an effective cleaning technique and ancillary analytical method for estimation of residues of selected kinase inhibitors from stainless steel and glass surfaces by swab sampling.

Importance of cleaning validation in the pharmaceutical industry cannot be overstated. It is essential for preventing cross-contamination, ensuring product quality & safety, and upholding regulatory standards. The present study involved development of an effective cleaning method for five selected kinase inhibitors binimetinib (BMT), selumetinib (SMT), brigatinib (BGT), capmatinib (CPT), and baricitinib (BRT). For checking the effectiveness of the developed cleaning technique, a sensitive and specific RP-HPLC based analytical method employing a diode array detector has been established to quantitate drug residue on glass and stainless steel surfaces. A reproducible swab sampling protocol utilizing TX714A Alpha swabs wetted with an extracting solvent has been developed to collect representative samples from both surfaces. Chromatographic separation of selected kinase inhibitors was achieved in gradient mode using an Agilent Zorbax eclipsed C18 column with acetonitrile and 10 mM ammonium formate as the mobile phase. The analytes were chromatographically separated in a 12 min run time. The mean swab recovery for each drug from glass and stainless steel surfaces exceeded 90%. Cleaning with IPA (70%) and acetone (70%) effectively removed residues for all five drugs. A solution comprising 10 mM SDS with 20% IPA demonstrated good efficacy in cleaning residues of BGT, BRT, and CPT, but exhibited lower efficacy for SMT and BMT.

LC/Q-TOF-MS-based structural characterization of enasidenib degradation products and establishment of a stability-indicating assay method: Insights into chemical stability.

RATIONALE: Enasidenib (EDB) is an orally active selective mutant isocitrate dehydrogenase-2 enzyme inhibitor approved by the U.S. Food and Drug Administration to treat acute myeloid leukemia. It lacks a reported forced degradation study and a stability-indicating assay method (SIAM). This study addresses this gap by establishing a degradation profile in accordance with the International Council for Harmonisation Q1A and Q1B (R2) guidelines and developing a validated SIAM for EDB. METHODS: EDB was exposed to forced degradation under various conditions (hydrolytic, photolytic, oxidative, and thermal stress). Degradation samples were analyzed using high-performance liquid chromatography on an Agilent ZORBAX Eclipse Plus C18 column with a mobile phase consisting of 0.1% formic acid in Milli-Q water and acetonitrile at a flow rate of 1 mL/min and detection at 270 nm. Liquid chromatography-quadrupole time-of-flight-high-resolution mass spectrometry (LC/Q-TOF HRMS) was used for the identification and characterization of degradation products. Nitrosamine risk assessment was conducted using a modified nitrosation assay procedure (NAP) test due to the presence of a secondary amine group in the drug, which is liable to forming nitrosamine drug substance-related impurities (NDSRI). RESULTS: The drug exhibited significant degradation under acidic, basic, photolytic, and oxidative conditions in the solution state. A total of nine degradation products (DP) were formed (DP-I, DP-III, and DP-IV: acidic conditions; DP-I and DP-III: basic conditions; DP-II, DP-V, DP-VI, and DP-VII: oxidative stress; and DP-VII, DP-VIII, and DP-IX: photolytic conditions), which were separated and identified using reversed-phase high-performance liquid chromatography and characterized using liquid chromatography-tandem mass spectrometry. The mechanism behind the formation of EDB degradation products has been discussed, and this study was the first to develop a degradation pathway for EDB. In addition, the possibilities of NDSRI formation for EDB were studied using a modified NAP test, which can contribute to the risk assessment of the drug. CONCLUSIONS: Forced degradation studies were conducted by establishing a SIAM for EDB. All the degradation products were characterized by mass spectral data obtained using LC/Q-TOF-HRMS.

Recent advancements in disulfide bridge characterization: Insights from mass spectrometry.

RATIONALE: Disulfide bridges (DSB) play an important role in stabilizing three-dimensional structures of biopharmaceuticals, single purified proteins, and various cyclic peptide drugs that contain disulfide in their structures. Incorrect cross-linking known as DSB scrambling results in misfolded structures that can be inactive, immunogenic, and susceptible to aggregation. Very few articles have been published on the experimental annotation of DSBs in proteins and cyclic peptide drugs. Accurate characterization of the disulfide bond is essential for understanding protein confirmation. METHODS: Characterizing DSBs using mass spectrometry (MS) involves the chemical and enzymatic digestion of samples to obtain smaller peptide fragments, in both reduced and nonreduced forms. Subsequently, these samples are analyzed using MS to locate the DSB, either through interpretation or by employing various software tools. RESULTS: The main challenge in DSB analysis methods using sample preparation is to obtain a sample solution in which nonnative DSBs are not formed due to high pH, temperature, and presence of free sulfhydryl groups. Formation of nonnative DSBs can lead to erroneous annotation of disulfide bond. Sample preparation techniques, fragmentation methods for DSB analysis, and contemporary approaches for DSB mapping using this fragmentation were discussed. CONCLUSIONS: This review presents the latest advancement in MS-based characterization; also a critical perspective is presented for further annotation of DSBs using MS, primarily for single purified proteins or peptides that are densely connected and rich in cysteine. Despite significant breakthroughs resulting from advancements in MS, the analysis of disulfide bonds is not straightforward; it necessitates expertise in sample preparation and interpretation.

Development of stability-indicating assay method and liquid chromatography-quadrupole-time-of-flight mass spectrometry-based structural characterization of the forced degradation products of alpelisib.

The US Food and Drug Administration and the European Medicines Agency approved alpelisib in 2019 for the treatment of metastatic breast cancer. A thorough literature review revealed that a stability-indicating analytical method (SIAM) is not available for the quantification of alpelisib and its degradation products (DPs). In this study, per the comprehensive stress study recommended by the International Council for Harmonisation (ICH), alpelisib was exposed to hydrolysis, oxidation, photolysis, and thermal stress. Degradation of the drug was observed under hydrolysis, oxidative, and photolysis conditions, whereas the drug was stable under thermal stress condition. We developed a SIAM for the separation of alpelisib and its major DPs that were formed under different stress conditions. The validation of the developed method was performed per ICH Q2(R1) guidelines. Five DPs were identified and characterized. Structure elucidation of all DPs was performed with the modern characterization tool of liquid chromatography-quadrupole time-of-flight mass spectrometer (LC-Q-TOF-MS/MS). The degradation pathway of the drug and its mechanisms were outlined, and in silico toxicity prediction was performed using the ProTox-II tool.

Identification and structural characterization of major stress degradation products of halcinonide by liquid chromatography-high-resolution mass spectrometry.

Halcinonide is a topical corticosteroid approved by the US Food and Drug Administration (FDA), known for its anti-inflammatory and antipruritic properties. The therapeutic benefits of halcinonide have rendered it an effective treatment regimen for various dermatological conditions such as psoriasis, dermatitis, and eczema. However, stability of the drug substance is a prerequisite in determining the therapeutic efficacy and plays a crucial role during formulation development and long-term storage. As corticosteroids are highly susceptible to degradation, the current study aims to expose halcinonide to different stress conditions and understand its stability behavior. An HPLC method was developed for the separation of halcinonide and its degradation products. Separation was accomplished in gradient mode using an Eclipse Plus C18 column (250 × 4.5 mm, 5 µm) with ammonium formate (10 mM, pH 6.5) and acetonitrile as the mobile phases. LC-Q-TOF/MS/MS studies were conducted on halcinonide, which led to the identification of degraded products using optimized mass parameters. A potential mechanistic degradation pathway for halcinonide, along with the major identified degradation products has been established. The chromatographic method that was developed has been validated in compliance with the Q2(R1) guideline of the International Council for Harmonization. ProTox-II was used to perform in silico toxicity studies in order to evaluate the toxicity potential of both halcinonide and the identified degradation products.

Recent Advances in Teriparatide Delivery by-virtue-of Novel Drug Delivery Approaches for the Management of Osteoporosis.

Osteoporosis is a bone incapacitating malady which globally accounts for over hundred million fractures annually. Therapeutic interventions for management of osteoporosis are divided as antiresorptive agents and osteoanabolic agents. Teriparatide is the only osteoana-bolic peptide which is available world-wide for the treatment of osteoporosis. It is administered as a daily subcutaneous injection for the treatment of osteoporosis which results in both poor patient compliance and increase in the cost of the therapy. Even after 20 years of clinical use of teriparatide, no formulation of teriparatide has yet been translated from lab to clinic which can be delivered by non-invasive route The present review critically discusses attempts made by the researchers for efficient delivery of teriparatide through various non-invasive routes such as oral, nasal, pulmonary, and transdermal route. It also discusses long-acting injectable formulations of teriparatide to improve patient compliance. Understanding on the pharmacology of teriparatide highlights the enhanced effectiveness of intermittent/pulsatile mode of teriparatide delivery which has also been elaborated. In addition, targeted delivery of teriparatide using different bone specific targeting moieties has been also discussed.

Production and characterization of bioactive peptides in fermented soybean meal produced using proteolytic Bacillus species isolated from kinema.

The study aims to enhance the functional properties of soybean meal (SBM) using potent proteolytic Bacillus strains isolated from kinema, a traditional fermented soybean product of Sikkim Himalaya. Selected Bacillus species; Bacillus licheniformis KN1G, B. amyloliquifaciens KN2G, B. subtilis KN36D, B. subtilis KN2B, and B. subtilis KN36D were employed for solid state fermentation (SSF) of SBM samples. The water and methanol extracts of SBM hydrolysates presented a significant increase in antioxidant activity. The water-soluble extracts of B. subtilis KN2B fermented SBM exhibited the best DPPH radical scavenging activity of 2.30 mg/mL. In contrast, the methanol-soluble extract of B. licheniformis KN1G fermented SBM showed scavenging activity of 0.51 mg/mL. Proteomic analysis of fermented soybean meal revealed several common and unique peptides produced by applying different starter cultures. Unique antioxidant peptides (HFDSEVVFF and VVDMNEGALFLPH) were identified from FSBM via LC/MS. B. subtilis KN36D showed the highest diversity of peptides produced during fermentation. The results indicate the importance of specific strains for fermentation to upgrade the nutritional value of raw fermented biomass.

Modified NAP test: A simple and Responsive Nitrosating Methodology for Risk Evaluation of NDSRIs.

N-Nitroso compounds have been listed as one of the cohorts of concern as per ICH M7. In recent years, the regulatory focus has shifted from common nitrosamines to nitroso-impurities of drug products. Thus, the detection and quantification of unacceptable levels of nitrosamine drug substance-related impurities are of great concern for analytical scientists during drug development. Moreover, risk assessment of nitrosamines is also an essential part of the regulatory filling. For risk assessment, the Nitrosation Assay Procedure suggested by WHO expert group in 1978 is being followed. However, it could not be adopted by the pharmaceutical industries due to the limitation of drug solubility and artefact formation in the test conditions. In this work, we have optimized an alternative nitrosation test to investigate the likelihood of direct nitrosation. The technique is simple, where the drug solubilized in an organic solvent is incubated at 37°C with a nitrosating agent named tertiary butyl nitrite in a 1:10 molar ratio. LC-UV/MS-based chromatographic method was developed to separate drug substances and respective nitrosamine impurities using the C18 analytical column. The methodology was successfully tested on five drugs with varying structural chemistry. The procedure is straightforward, effective, and quick for the nitrosation of secondary amines. This modified nitrosation test and WHO prescribed nitrosation test have been compared and found that the modified methodology is more effective and time-saving.

Recurrent Pericarditis: A Rare Adverse Effect of Mesalamine.

Inflammation of the myocardium (myocarditis), pericardium (pericarditis), or both (myopericarditis) is a rare but potentially lethal side effect of mesalamine, a drug widely used in the treatment of inflammatory bowel disease (IBD). A 64-year-old female with a history of ulcerative colitis on mesalamine presented with dyspnea and chest pain. The patient was found to have mild to moderate pericardial effusion with signs of pericarditis. Further workup revealed positive rheumatoid factor, antineutrophil cytoplasmic autoantibody, cytoplasmic (c-ANCA), and antinuclear antibodies (ANA), raising suspicion for rheumatoid-associated pericarditis. She was discharged with a prednisone taper and an outpatient rheumatology follow-up. However, the patient presented again in three months with similar complaints and was found to have recurrent pericarditis. On this admission, mesalamine-induced pericarditis was considered in the differential diagnosis, and it was stopped on discharge. On a three-month follow-up, the patient showed complete resolution. Mesalamine-induced pericarditis is a rare side effect of this drug, and prompt recognition and appropriate intervention are important to prevent the progression of the inflammation and avoid adverse cardiovascular outcomes. The association of IBD with extra-intestinal cardiac manifestations can delay early diagnosis and treatment.

Genome-resolved metagenomics inferred novel insights into the microbial community, metabolic pathways, and biomining potential of Malanjkhand acidic copper mine tailings.

Mine tailing sites provide profound opportunities to elucidate the microbial mechanisms involved in ecosystem functioning. In the present study, metagenomic analysis of dumping soil and adjacent pond around India's largest copper mine at Malanjkhand has been done. Taxonomic analysis deciphered the abundance of phyla Proteobacteria, Bacteroidetes, Acidobacteria, and Chloroflexi. Genomic signatures of viruses were predicted in the soil metagenome, whereas Archaea and Eukaryotes were noticed in water samples. Mesophilic chemolithotrophs, such as Acidobacteria bacterium, Chloroflexi bacterium, and Verrucomicrobia bacterium, were predominant in soil, whereas, in the water sample, the abundance of Methylobacterium mesophilicum, Pedobacter sp., and Thaumarchaeota archaeon was determined. The functional potential analysis highlighted the abundance of genes related to sulfur, nitrogen, methane, ferrous oxidation, carbon fixation, and carbohydrate metabolisms. The genes for copper, iron, arsenic, mercury, chromium, tellurium, hydrogen peroxide, and selenium resistance were found to be predominant in the metagenomes. Metagenome-assembled genomes (MAGs) were constructed from the sequencing data, indicating novel microbial species genetically related to the phylum predicted through whole genome metagenomics. Phylogenetic analysis, genome annotations, functional potential, and resistome analysis showed the resemblance of assembled novel MAGs with traditional organisms used in bioremediation and biomining applications. Microorganisms harboring adaptive mechanisms, such as detoxification, hydroxyl radical scavenging, and heavy metal resistance, could be the potent benefactions for their utility as bioleaching agents. The genetic information produced in the present investigation provides a foundation for pursuing and understanding the molecular aspects of bioleaching and bioremediation applications.

Cutting-edge strategies and critical advancements in characterization and quantification of metabolites concerning translational metabolomics.

Despite remarkable progress in drug discovery strategies, significant challenges are still remaining in translating new insights into clinical applications. Scientists are devising creative approaches to bridge the gap between scientific and translational research. Metabolomics is a unique field among other omics techniques for identifying novel metabolites and biomarkers. Fortunately, characterization and quantification of metabolites are becoming faster due to the progress in the field of orthogonal analytical techniques. This review detailed the advancement in the progress of sample preparation, and data processing techniques including data mining tools, database, and their quality control (QC). Advances in data processing tools make it easier to acquire unbiased data that includes a diverse set of metabolites. In addition, novel breakthroughs including, miniaturization as well as their integration with other devices, metabolite array technology, and crystalline sponge-based method have led to faster, more efficient, cost-effective, and holistic metabolomic analysis. The use of cutting-edge techniques to identify the human metabolite, including biomarkers has proven to be advantageous in terms of early disease identification, tracking the progression of illness, and possibility of personalized treatments. This review addressed the constraints of current metabolomics research, which are impeding the facilitation of translation of research from bench to bedside. Nevertheless, the possible way out from such constraints and future direction of translational metabolomics has been conferred.

Presentation and outcome of myocardial infarction with non-obstructive coronary arteries in coronavirus disease 2019.

Among the cardiac complications of coronavirus disease 2019 (COVID-19), one increasingly reported in the literature is myocardial infarction with non-obstructive coronaries (MINOCA). We reviewed all reported cases of MINOCA in COVID-19 patients to summarize its clinical features, evaluation, and treatment. We performed a literature search in Pubmed using the search terms 'COVID-19' and 'MINOCA' or 'non-obstructive coronaries'. Among the reported cases, the mean age was 61.5 years (SD ± 13.4), and 50% were men. Chest pain was the presenting symptom in five patients (62.5%), and hypertension was the most common comorbidity (62.5%). ST-elevation was seen in most patients (87.5%), and the overall mortality rate was 37.5%. MINOCA in COVID-19 is an entity with a broad differential diagnosis. Therefore, a uniform algorithm is needed in its evaluation to ensure timely diagnosis and management.

Role of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in cryoballoon ablation outcomes for paroxysmal atrial fibrillation.

BACKGROUND: Cryoballoon ablation (CBA) is recommended for patients with paroxysmal atrial fibrillation (AF) refractory to antiarrhythmic drugs. However, only 80% of patients benefit from initial CBA. There is growing evidence that pretreatment with angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) decreases the recurrence of AF postablation, particularly in nonparoxysmal AF undergoing radiofrequency ablation. The role of ACEIs and ARBs in patients with paroxysmal AF in CBA remains unknown. We decided to investigate the role of ACEIs and ARBs in preventing the recurrence of atrial arrhythmia (AA) following CBA for paroxysmal AF. AIM: To investigate the role of ACEIs and ARBs in preventing recurrence of AA following CBA for paroxysmal AF. METHODS: We followed 103 patients (age 60.6 ± 9.1 years, 29% women) with paroxysmal AF undergoing CBA 1-year post procedure. Recurrence was assessed by documented AA on electrocardiogram or any form of long-term cardiac rhythm monitoring. A multivariable Cox proportional hazard model was used to assess if ACEI or ARB treatment predicted the risk of AA recurrence. RESULTS: After a 1-year follow-up, 19 (18.4%) participants developed recurrence of AA. Use of ACEI or ARB therapy was noted in the study population. Patients on ACEI/ARB had a greater prevalence of hypertension and coronary artery disease. On a multivariate model adjusted for baseline demographics and risk factors for AF, ACEI or ARB therapy did not prevent recurrence of AA following CBA (P = 0.72). Similarly, on Kaplan-Meier analysis pretreatment with ACEI/ARB did not predict the time to first recurrence of AA (P = 0.2173). CONCLUSION: In our study population, preablation treatment with an ACEI or ARB had no influence on the recurrence of AA following CBA for paroxysmal AF.

Brugada Sign in a Cardiac Transplant Donor.

Brugada syndrome (BrS) is a rare entity represented by the Brugada sign on an electrocardiogram (EKG) and is associated with sudden cardiac death (SCD). There is little data to guide the management of donor Brugada syndrome in the setting of cardiac transplantation. A 31-year-old male sustained out-of-hospital cardiac arrest secondary to polysubstance use and was found asystole. Bystander cardiopulmonary resuscitation (CPR) with advanced cardiovascular life support (ACLS) protocol was initiated. Return of spontaneous circulation (ROSC) was achieved and the patient was taken to the emergency room (ER) in sinus rhythm with an initial presenting EKG showing the Brugada sign. A toxicological screen for cocaine was positive. The patient was eventually declared brain dead and underwent angiographic and echocardiographic evaluation as a donor heart for cardiac transplantation and was accepted for transplantation. Cardiac arrest in a young patient with a Brugada sign on EKG is a concern for BrS. Cocaine exerts a sodium channel blockade that can unmask BrS. Genetic testing for sodium voltage-gated channel alpha subunit 5 (​​​​​​SCN5A) gene mutation was negative, however, only 15% to 30% of patients carry the mutation. We proceeded with cardiac transplantation and suggested an implantable cardioverter defibrillator (ICD) for primary prevention in the recipient, should further specialized testing reveal a continued concern for BrS. We suggest the necessity for further data to guide decisions in patients with BrS undergoing cardiac transplantation.

Metagenomic views on taxonomic and functional profiles of the Himalayan Tsomgo cold lake and unveiling its deterzome potential.

Cold habitat is considered a potential source for detergent industry enzymes. This study aims at the metagenomic investigation of Tsomgo lake for taxonomic and functional annotation, unveiling the deterzome potential of the residing microbiota at this site. The present investigation revealed molecular profiling of microbial community structure and functional potential of the high-altitude Tsomgo lake samples of two different temperatures, harvested during March and August. Bacteria were found to be the most dominant phyla, with traces of genomic pieces of evidence belonging to archaea, viruses, and eukaryotes. Proteobacteria and Actinobacteria were noted to be the most abundant bacterial phyla in the cold lake. In-depth metagenomic investigation of the cold aquatic habitat revealed novel genes encoding detergent enzymes, amylase, protease, and lipase. Further, metagenome-assembled genomes (MAGs) belonging to the psychrophilic bacterium, Arthrobacter alpinus, were constructed from the metagenomic data. The annotation depicted the presence of detergent enzymes and genes for low-temperature adaptation in Arthrobacter alpinus. Psychrophilic microbial isolates were screened for lipase, protease, and amylase activities to further strengthen the metagenomic findings. A novel strain of Acinetobacter sp. was identified with the dual enzymatic activity of protease and amylase. The bacterial isolates exhibited hydrolyzing activity at low temperatures. This metagenomic study divulged novel genomic resources for detergent industry enzymes, and the bacterial isolates secreting cold-active amylase, lipase, and protease enzymes. The findings manifest that Tsomgo lake is a potential bioresource of cold-active enzymes, vital for various industrial applications.

From Takotsubo to Yamaguchi.

Our patient was a 56-year-old Caucasian female who had 34 emergency department visits to our center with recurrent chest pain, of which eleven were of cardiac etiology, involving cardiac causes over the period of seven years. Her chest pain was diagnosed as atypical during her previous visits. Chest CT revealed "ace-of-spades" in the cardiac transverse section. A transthoracic echocardiogram (TTE) demonstrated apical hypertrophy with end-systolic cavity obliteration and an ejection fraction (EF) of 65%-70%, seated amidst a normal-sized left ventricle, with normal wall thickness, indicating Yamaguchi syndrome. In the case report, we portray the need to widen the spectrum of differentials in an encounter with a patient presenting with chest pain.