Environment dependent expression of mycobacterium hormone sensitive lipases: expression pattern under ex-vivo and individual in-vitro stress conditions in M. tuberculosis H37Ra.

BACKGROUND: Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kinds of lipids. In comparison to single human Hormone Sensitive Lipase (hHSL), that is induced under nutritional stress, twelve serine hydrolases are annotated as HSL in Mycobacterium tuberculosis (mHSL). Mycobacterium is exposed to multiple stresses inside the host. Therefore, the present study was carried out to investigate if mHSL are also expressed under stress condition and if there is any correlation between various stress conditions and expression pattern of mHSL. METHODS AND RESULTS: The expression pattern of mHSL under different environmental conditions (in-vitro and ex-vivo) were studied using qRT-PCR in M. tuberculosis H37Ra strain with 16 S rRNA as internal control. Out of 12, only two genes (lipU and lipY) were expressed at very low level in mid log phase culture under aerobic conditions, while 9 genes were expressed at stationary phase of growth. Ten mHSLs were expressed post-infection under ex-vivo conditions in time dependent manner. LipH and lipQ did not express at any time point under ex-vivo condition. The relative expression of most of the genes under individual stress was much higher than observed in ex-vivo conditions. The expression pattern of genes varied with change in stress condition. CONCLUSIONS: Different sets of mHSL genes were expressed under different individual stress conditions pointing towards the requirement of different mHSL to combat different stress conditions. Overall, most of the mHSLs have demonstrated stress dependent expression pointing towards their role in intracellular survival of mycobacteria.

Cereal starch nanoparticles-A prospective food additive: A review.

Starch is one of the most abundant biopolymers in nature and is typically isolated from plants in the form of micro-scale granules. Raw starch has limited applications due to its innate disadvantages such as poor solubility in cold water, tendency to retrograde and high viscosity once it is gelatinized. Therefore, some degree of modification is required to enhance its functionality. Starch nanoparticle is one of the products of such modification. Chemical, enzymatic, and physical treatments are used for the preparation of starch nanoparticles and to study their granular and molecular structures. Characterization of starch nanoparticles on the size distribution, crystalline structure, and physical properties in relation to the starch sources and preparation methods can be done using various characterization tools e.g. Scanning Electron Microscopy, Transmission Electron Microscopy, Atomic Florescence Microscopy, etc. Starch nanoparticles can be used as a food additive as it has adverse range of uses in food such as emulsion stabilizer, fat replacer, Thickener, or rheology modifier etc.

Suicidal Ideation in the Setting of Neurocysticercosis.

Neurocysticercosis (NCC) is considered a significant health concern in developing countries in parts of Asia, Africa, and Central and South America. However, with the increased immigration, it is now becoming increasingly prevalent in the United States. NCC has psychiatric implications often neglected and not recognized in the initial diagnostic workup of patients from developing countries suffering from seizures and psychiatric illnesses, such as depression. This case report aims to signify the presentation of NCC and illustrate the importance of the psychiatric manifestations of NCC in patients. We discuss the case of a 32-year-old female patient from a rural town in Central America who immigrated to New York and presented with uncontrolled seizures and symptomatic depression with suicidal ideations.

Immunotherapy guided precision medicine in solid tumors.

Cancer is no longer recognized as a single disease but a collection of diseases each with its defining characteristics and behavior. Even within the same cancer type, there can be substantial heterogeneity at the molecular level. Cancer cells often accumulate various genetic mutations and epigenetic alterations over time, leading to a coexistence of distinct subpopulations of cells within the tumor. This tumor heterogeneity arises not only due to clonal outgrowth of cells with genetic mutations, but also due to interactions of tumor cells with the tumor microenvironment (TME). The latter is a dynamic ecosystem that includes cancer cells, immune cells, fibroblasts, endothelial cells, stromal cells, blood vessels, and extracellular matrix components, tumor-associated macrophages and secreted molecules. The complex interplay between tumor heterogeneity and the TME makes it difficult to develop one-size-fits-all treatments and is often the cause of therapeutic failure and resistance in solid cancers. Technological advances in the post-genomic era have given us cues regarding spatial and temporal tumor heterogeneity. Armed with this knowledge, oncologists are trying to target the unique genomic, epigenetic, and molecular landscape in the tumor cell that causes its oncogenic transformation in a particular patient. This has ushered in the era of personalized precision medicine (PPM). Immunotherapy, on the other hand, involves leveraging the body's immune system to recognize and attack cancer cells and spare healthy cells from the damage induced by radiation and chemotherapy. Combining PPM and immunotherapy represents a paradigm shift in cancer treatment and has emerged as a promising treatment modality for several solid cancers. In this chapter, we summarise major types of cancer immunotherapy and discuss how they are being used for precision medicine in different solid tumors.

Closed-Loop Gastric Outlet Obstruction Secondary to Duodenal Ulcer in a Patient With Esophageal Stricture.

Closed-loop gastric outlet obstruction (GOO) is a rare complication that results from a mechanical obstruction in the pylorus or duodenum. In the early 1990s, the common cause of GOO was peptic ulcer disease, accounting for 5% to 10% of hospital admissions. Peptic ulcer disease is the disruption of the mucosal integrity in the stomach and duodenum and can be categorized into gastric ulcers and duodenal ulcers. With the treatment for Helicobacter  pylori and the increased use of proton pump inhibitors (PPI), GOO now occurs in fewer than 5% of patients with duodenal ulcer disease and even less in those with gastric ulcer disease. Although the morbidity of duodenal ulcers has been declining in recent years, the incidence of post-bulbar duodenal ulcer (PBDU) remains at a constant 9.33%, primarily due to diagnostic and therapeutic difficulties. Additionally, fewer than 5% of obstructing duodenal ulcers are caused by PBDU, and even fewer are located in the second or third portions of the duodenum. Ulcers located in the distal part of the duodenum raise concern for syndromes associated with hypersecretion of acid, including Zollinger-Ellison syndrome (ZES). The ZES is rare, accounting only for 0.1% of all duodenal ulcers. Here, we present a case where a patient with esophageal stricture developed a rare case of closed-loop GOO secondary to a duodenal ulcer. The patient, initially treated for esophageal perforation, developed an esophageal stricture. The patient was being worked up for ZES and multiple endocrine neoplasia link type 1 (MEN1) syndrome due to his concerning laboratory findings and rare clinical presentation.

Phenolic Biotransformations in Wheatgrass Juice after Primary and Secondary Fermentation.

Fermented wheatgrass juice was prepared using a two-stage fermentation process by employing Saccharomyces cerevisiae and recombinant Pediococcus acidilactici BD16 (alaD+). During fermentation, a reddish-brown hue appeared in wheatgrass juice due to production of different types of red pigments. The fermented wheatgrass juice has considerably higher content of anthocyanins, total phenols and beta-carotenes as compared to unfermented wheatgrass juice. It has low ethanol content, which might be ascribed to the presence of certain phytolignans in wheatgrass juice. Several yeast-mediated phenolic transformations (such as bioconversion of coumaric acid, hydroxybenzoic acid, hydroxycinnamic acid and quinic acid into respective derivatives; glycosylation and prenylation of flavonoids; glycosylation of lignans; sulphonation of phenols; synthesis of carotenoids, diarylnonanoids, flavanones, stilbenes, steroids, quinolones, di- and tri-terpenoids and tannin) were identified in fermented wheatgrass juice using an untargeted liquid chromatography (LC)-mass spectrometry (MS)-matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)/time-of-flight (TOF) technique. The recombinant P. acidilactici BD16 (alaD+) also supported flavonoid and lignin glycosylation; benzoic acid, hydroxycoumaric acid and quinic acid derivatization; and synthesis of anthraquinones, sterols and triterpenes with therapeutic benefits. The information presented in this manuscript may be utilized to elucidate the importance of Saccharomyces cerevisiae and P. acidilactici BD16 (alaD+) mediated phenolic biotransformations in developing functional food supplements such as fermented wheatgrass juice.

Effect of extrusion processing on techno-functional properties, textural properties, antioxidant activities, in vitro nutrient digestibility and glycemic index of sorghum-chickpea-based extruded snacks.

Physico-chemical, textural, functional, and nutritional properties of the twin screw extruded whole sorghum-chickpea (8:2) snacks was investigated using in vitro procedures. The extruded snacks were analyzed for the effect of variations in extruded conditions on their properties: barrel BT (BT) (130-170°C) and feed moisture (FM) (14%-18%), keeping screw speed constant (400 rpm). The results revealed that specific mechanical energy (SME) decreased (74.4-60.0) in response to rise in both BT and FM, whereas expansion ratio (ER) had shown an alternative relation as it decreased with elevated FM (2.17 at 14%, 130°C to 2.14 at 16%, 130°C) and increased with BT (1.75 at 18%, 130°C to 2.48 at 18%, 170°C). The values of WAI and WSI improved with the surge in BT, which was associated with enhanced disruption of starch granules at higher BT. Raise in FM incremented the total phenolic content (TPC) and hence the antioxidant activity (AA) (FRAP and DPPH) along with the hardness of snacks. As per in vitro starch digestibility is concerned, slowly digestible starch (SDS) content as well as glycemic index (51-53) of the extrudates depressed with increasing BT and FM. Also, lower BT and FM improved the functional properties such as expansion ratio, in-vitro protein digestibility, and overall acceptability of the snacks. A positive correlation was seen among SME and hardness of the snacks, WSI and ER, TPC and AA, SDS and Exp-GI, color and OA, texture and OA.

Brain Abscess Caused by Nocardia farcinica in a Young Immunocompetent Patient.

Cerebral nocardiosis is a rare opportunistic infectious disease that occurs mainly in immunocompromised hosts; however, immunocompetent patients may be affected too. It often results in the formation of intraparenchymal brain abscess, which represents only 2% of all cerebral abscesses. The overall mortality rate exceeds 20% in immunocompetent patients and 55% in immunocompromised patients. Bacteriological diagnosis is often confirmed only after the surgical excision of the abscess. Thus, the initiation of effective therapy is frequently delayed. Our goal is to highlight a diagnostic approach to cerebral nocardiosis in an immunocompetent patient with the purpose of accelerating the initiation of the appropriate therapy. We report a rare case of brain abscess caused by Nocardia farcinica in a 39-year-old male, a resident of New York City, USA, with a past medical history of intravenous (IV) drug use, who was admitted for altered mental status. The patient was cachectic and ill-appearing. Initial laboratory tests showed neutrophilic leukocytosis. Computed tomography (CT) of the head revealed a large ill-defined multilobulated mass of size 6 × 5 × 4.5 cm in the right cerebral hemisphere, which was confirmed with magnetic resonance imaging (MRI). The hospital course was complicated by the deterioration of mental status requiring endotracheal intubation. The patient underwent a right-sided hemicraniectomy; a wound culture identified Nocardia farcinica. The patient was started on intravenous (IV) Bactrim, which caused an allergic reaction. Thus, he was switched to IV imipenem-cilastatin. After E-test was performed, the patient was switched to oral linezolid. The initiation of targeted antibiotic therapy was crucial for the management of this patient and resulted in a good clinical outcome. In conclusion, cerebral nocardiosis, being an unusual and a potentially fatal infection, should be considered in the differential diagnosis of brain abscess even in immunocompetent hosts. Prompt bacteriological diagnosis helps to initiate a specific antimicrobial therapy. Long-term antimicrobial therapy and long-term follow-up are necessary to prevent relapse.

Pathogenesis of Alzheimer's Disease and Diversity of 1,2,3-Triazole Scaffold in Drug Development: Design Strategies, Structural Insights, and Therapeutic Potential.

Alzheimer's disease is a most prevalent form of dementia all around the globe and currently poses a significant challenge to the healthcare system. Currently available drugs only slow the progression of this disease rather than provide proper containment. Identification of multiple targets responsible for this disease in the last three decades established it as a multifactorial neurodegenerative disorder that needs novel multifunctional agents for its management and the possible reason for the failure of currently available single target clinical drugs. 1,2,3-Triazole is a miraculous nucleus in medicinal chemistry and the first choice for development of multifunctional hybrid molecules. Apart from that, it is an integral component of various drugs in clinical trials as well as in clinical practice. This review is focused on the pathogenesis of Alzheimer's disease and 1,2,3-triazole containing derivatives developed in recent decades as potential anti-Alzheimer's agents. The review will provide (A) precise insight of various established targets of Alzheimer's disease including cholinergic, amyloid, tau, monoamine oxidases, glutamate, calcium, and reactive oxygen species hypothesis and (B) design hypothesis, structure-activity relationships, and pharmacological outcomes of 1,2,3-triazole containing multifunctional anti-Alzheimer's agents. This review will provide a baseline for various research groups working on Alzheimer's drug development in designing potent, safer, and effective multifunctional anti-Alzheimer's candidates of the future.

The lipolytic activity of LipJ, a stress-induced enzyme, is regulated by its C-terminal adenylate cyclase domain.

Aim: The confirmation of lipolytic activity and role of Rv1900c in the Mycobacterium physiology Methods:rv1900c/N-terminus domain (rv1900NT) were cloned in pET28a/Escherichia coli, purified by affinity chromatography and characterized. Results: A zone of clearance on tributyrin-agar and activity with pNP-decanoate confirmed the lipolytic activity of Rv1900c. The Rv1900NT demonstrated higher enzyme specific activity, Vmax and kcat, but Rv1900c was more thermostable. The lipolytic activity of Rv1900c decreased in presence of ATP. Mycobacterium smegmatis expressed rv1900c/rv1900NT-altered colony morphology, growth, cell surface properties and survival under stress conditions. The effect was more prominent with Rv1900NT as compared with Rv1900c. Conclusion: The study confirmed the lipolytic activity of Rv1900c and suggested its regulation by the adenylate cyclase domain and role in the intracellular survival of bacteria.

Lay abstract Tuberculosis (TB) remains the top contagious/infectious killer in the world. It is caused by the bacteria Mycobacterium tuberculosis. The bacteria resides/replicates in the immune cell that normally has to eradicate infectious microorganisms. Though the treatment of TB is available, the emergence of drug-resistant bacteria is of major concern. The treatment of drug-resistant TB has been reported to be more difficult due to lengthy and complex treatment regimens. Therefore, there is an urgent need for new and better drugs to treat TB/drug-resistant TB. For this purpose understanding the role of each protein in the physiology of mycobacteria is required. Lipids play a critical role in the intracellular survival of this pathogen in the host. Our study demonstrated that LipJ supported the intracellular survival of bacteria. Therefore, it could be a potential drug target.

Rv0518, a nutritive stress inducible GDSL lipase of Mycobacterium tuberculosis, enhanced intracellular survival of bacteria by cell wall modulation.

Rv0518, a hypothetical protein of Mycobacterium tuberculosis, was designated as possible exported protein. In-silico analysis suggested that the protein belonged to the family of GDSL lipases. In this study, rv0518 gene was cloned and expressed in E. coli followed by purification and characterization. It possessed lipolytic activity, preferably hydrolyzed pNP-decanoate at pH 9.0 and 40 °C. The enzyme was stable till 50 °C and wider pH range (5.0-11.0). The predicted active site residues, Ser-46, Asp-205, His-208, Gly-87, Asn-120 were confirmed by site directed mutagenesis. rv0518 gene expression in M. tuberculosis H37Ra was up-regulated under nutrient starvation and the protein was detected in membrane fraction. The expression of rv0518 in M. smegmatis altered the colony morphology/growth kinetics, provided resistance to SDS, lysozyme, anti-TB drugs and enhanced in vitro survival of M. smegmatis under nutritive stress. The total lipid content and trehalose dimycolate was elevated in M. smegmatis expressing rv0518 gene. The presence of Rv0518 enhanced infection ability and intracellular survival capability of M. smegmatis. Hence, Rv0518 is a cell wall associated GDSL lipase might helped bacteria to utilize glycerol/lipids for its growth as well as provided resistance to various intracellular stresses by cell wall modulation resulting in its enhanced intracellular survival.

Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements.

E. coli is most preferred system used for the production of recombinant proteins in bacteria and the availability of improved genetic tools/methods are making it more valuable than ever. Major challenges faced by this expression system are the expression of unusually difficult/complex proteins with rare codons or membrane and toxic proteins. The proteins expressed either in large amount or hydrophobic in nature tend to form insoluble mass. Despite the appropriate expression system, some proteins express at very low level or not at all. Choosing the correct expression system/protocols are obligatory for the substantial expression of protein in the native form. A number of vectors, their compatible hosts and culture conditions can be used to express recombinant proteins in large amounts and in native form. Also, vectors with the fusion tags/chaperons facilitate protein expression in soluble fraction and assist in proper protein folding besides restoring the native structure of protein. The recovery of native proteins from insoluble inclusion bodies can be achieved by optimization of refolding conditions. In the present review, we discussed recent updates on prokaryotic expression system for successful heterologous gene expression in E. coli and focused on strategies to maximize the yields of native recombinant proteins.

Rv1288, a Two Domain, Cell Wall Anchored, Nutrient Stress Inducible Carboxyl-Esterase of Mycobacterium tuberculosis, Modulates Cell Wall Lipid.

Rv1288, a conserved hypothetical protein of M. tuberculosis (M.tb), was recently characterized as two-domain esterase enzyme by in silico study. In the present study, Rv1288 and its domains (Est and Lyt) were cloned individually from M.tb into E. coli for expression and purification. The purified rRv1288 and rEst proteins exhibited lipolytic activity with medium chain length esters as optimum substrates, while Lyt domain did not show enzymatic activity. However, presence of Lyt domain resulted in enhanced rate of protein aggregation at higher temperature. Both rRv1288 and rEst followed the similar patterns of substrate specificity, temperature and pH activity. Site directed mutagenesis confirmed the Ser-294, Asp-391 and His-425 as catalytic site residues. Rv1288 was found to be present in cell wall fraction of M.tb H37Ra. Peptidoglycan binding activity of Rv1288 and its domains demonstrated that the Lyt domain is essential for anchoring protein to the cell wall. Expression of rv1288 was up regulated in M.tb under nutrient starved condition. Over expression of rv1288 in surrogate host M. smegmatis led to change in colony morphology, enhanced pellicle and aggregate formation that might be linked with the changed lipid composition of bacterial cell wall. Cell wall of M. smegmatis expressing rv1288 had higher amount of lipids, with a significant increase in trehalose dimycolate content. Rv1288 also leads to increase in drug resistance of M. smegmatis. Rv1288 also enhanced the intracellular survival of M. smegmatis in Raw264.7 cell line. Overall, this study suggested that Rv1288, a cell wall localized carboxyl hydrolase with mycolyl-transferase activity, modulated the cell wall lipids to favor the survival of bacteria under stress condition.

mbtJ: an iron stress-induced acetyl hydrolase/esterase of Mycobacterium tuberculosis helps bacteria to survive during iron stress.

AIM: mbtJ from Mycobacterium tuberculosis H37Rv is a member of mbt A-J operon required for mycobactin biogenesis. MATERIALS & METHODS: The esterase/acetyl-hydrolase activity of mbtJ was determined by pNP-esters/native-PAGE and expression under iron stress by quantitative-PCR. Effect of gene on growth/survival of Mycobacterium was studied using antisense. Its effect on morphology, growth/infection was studied in Mycobacterium smegmatis. RESULTS: It showed acetyl hydrolase/esterase activity at pH 8.0 and 50°C with pNP-butyrate. Its expression was upregulated under iron stress. The antisense inhibited the survival of bacterium during iron stress. Expression of mbtJ changed colony morphology and enhanced the growth/infection in M. smegmatis. CONCLUSION: mbtJ, an acetyl-hydrolase/esterase, enhanced the survival of M. tuberculosis under iron stress, affected the growth/infection efficiency in M. smegmatis, suggesting its pivotal role in the intracellular survival of bacterium.