Hump-Nosed Pit Viper (Hypnale hypnale) Venom-Induced Irreversible Red Blood Cell Aggregation, Inhibition by Monovalent Anti-Venom and N-Acetylcysteine.

Envenomation by the Hypnale hypnale in the Western Ghats of India (particularly in the Malabar region of Kerala) and the subcontinent island nation of Sri Lanka is known to inflict devastating mortality and morbidity. Currently, H. hypnale bites in India are devoid of anti-venom regimens. A detailed characterization of the venom is essential to stress the need for therapeutic anti-venom. Notably, the deleterious effects of this venom on human blood cells have largely remained less explored. Therefore, in continuation of our previous study, in the present study, we envisioned investigating the effect of venom on the morphological and physiological properties of red blood cells (RBCs). The venom readily induced deleterious morphological changes and, finally, the aggregation of washed RBCs. The aggregation process was independent of the ROS and the intracellular Ca2+ ion concentration. Confocal and scanning electron microscopy (SEM) images revealed the loss of biconcave morphology and massive cytoskeletal disarray. Crenation or serrated plasma membrane projections were evenly distributed on the surface of the RBCs. The venom did not cause the formation of methemoglobin in washed RBCs but was significantly induced in whole blood. Venom did not affect glucose uptake and Na+/K+ -ATPase activity but inhibited glucose 6 phosphate dehydrogenase activity and decreased the fluidity of the plasma membrane. Venom-induced RBC aggregates exhibited pro-coagulant activity but without affecting platelet aggregation. In pre-incubation or co-treatment studies, none of the bioactive compounds, such as melatonin, curcumin, fisetin, berberine, and quercetin, sugars such as mannose and galactose, and therapeutic polyvalent anti-venoms (Bharat and VINS) were inhibited, whereas only N-acetylcysteine and H. hypnale monovalent anti-venom could inhibit venom-induced deleterious morphological changes and aggregation of RBCs. In post-treatment studies, paradoxically, none of the bioactives and anti-venoms, including N-acetylcysteine and H. hypnale monovalent anti-venom, reversed the venom-induced RBC aggregates.

Inhibitory and anti-adherent effects of Piper betle L. leaf extract against Acanthamoeba triangularis in co-infection with Staphylococcus aureus and Pseudomonas aeruginosa: A sustainable one-health approach.

Background and Aim: Keratitis is a serious ocular infection often caused by pathogenic microorganisms such as Acanthamoeba spp. Among other harmful microbes, Acanthamoeba keratitis presents a particular challenge due to its resistance to conventional antimicrobial agents. Piper betle Linn., commonly known as betel leaf, has been traditionally used for its medicinal properties. This study aimed to assess the potential of the leaf ethanol extract of P. betle Linn. in the treatment of Acanthamoeba triangularis in monoculture and co-culture with two prevalent pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, associated with keratitis. Materials and Methods: Minimum inhibitory concentrations (MICs) of A. triangularis, S. aureus, and P. aeruginosa extracts in monoculture and coinfected conditions were examined. In addition, this study explored the potential of the extract in preventing Acanthamoeba adherence in both monoculture and co-culture environments. Scanning electron microscopy (SEM) analysis confirmed the impact of the extract on Acanthamoeba cell membranes, including acanthopodia. Furthermore, a time-kill kinetic assay was used to validate the amoebicidal activity of the extract against A. triangularis and the tested bacteria. Results: MICs for trophozoites, cysts, P. aeruginosa, and S. aureus in the monoculture were 0.25, 0.25, 0.51, and 0.128 mg/mL, respectively, whereas the MICs for Acanthamoeba coinfected with bacteria were higher than those in the monoculture. This extract inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. Moreover, P. betle extract effectively prevented the adherence of Acanthamoeba to contact lenses under monoculture conditions. SEM analysis confirmed that P. betle extract affects the cell membrane of Acanthamoeba, including Acanthopodia. In addition, the time-kill kinetic assay confirmed that the extract contained amoebicidal activity against A. triangularis, including the tested bacteria. Notably, S. aureus was more susceptible than A. triangularis and P. aeruginosa to P. betle extract treatment. Unexpectedly, our study revealed that S. aureus negatively affected A. triangularis in the co-culture after 3 days of incubation, whereas P. aeruginosa facilitated the growth of A. triangularis in the presence of the extract. Conclusion: This study provides compelling evidence of the anti-adhesive and anti-Acanthamoeba properties of P. betle leaf extract against A. triangularis under monoculture and co-culture conditions. The observed impact on Acanthamoeba cell membranes, coupled with the time-kill kinetic assay results, underscores the potential of P. betle leaf extract as a promising agent for combating Acanthamoeba-related infections in humans and animals.

Oxidative Stress-Induced Platelet Apoptosis/Activation: Alleviation by Purified Curcumin via ASK1-JNK/p-38 Pathway.

Platelets are known for their indispensable role in hemostasis and thrombosis. However, alteration in platelet function due to oxidative stress is known to mediate various health complications, including cardiovascular diseases and other health complications. To date, several synthetic molecules have displayed antiplatelet activity; however, their uses are associated with bleeding and other adverse effects. The commercially available curcumin is generally a mixture of three curcuminoids: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Although crude curcumin is known to inhibit platelet aggregation, the effect of purified curcumin on platelet apoptosis, activation, and aggregation remains unclear. Therefore, in this study, curcumin was purified from a crude curcumin mixture and the effects of this preparation on the oxidative stress-induced platelet apoptosis and activation was evaluated. 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) compound was used as an inducer of oxidative stress. Purified curcumin restored AAPH-induced platelet apoptotic markers like reactive oxygen species, intracellular calcium level, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release from mitochondria to the cytosol, and phosphatidyl serine externalization. Further, it inhibited the agonist-induced platelet activation and aggregation, demonstrating its antiplatelet activity. Western blot analysis confirms protective effect of the purified curcumin against oxidative stress-induced platelet apoptosis and activation via downregulation of MAPKs protein activation, including ASK1, JNK, and p-38. Together, these results suggest that the purified curcumin could be a potential therapeutic bioactive molecule to treat the oxidative stress-induced platelet activation, apoptosis, and associated complications.

Fluorodeoxyglucose-positron emission tomography (FDG-PET) of carotid arteries and non-alcoholic fatty liver disease (NAFLD): An analytical cross-sectional study from a teaching hospital, Kerala, South India.

Introduction: Evidence related to carotid artery F-fluorodeoxyglucose-positron emission tomography (FDG -PET) and non-alcoholic fatty liver disease (NAFLD) is limited from a low-resource setting. The present study aims to examine the association between FDG-PET uptakes by the carotid arteries in patients having different grades of NAFLD. Materials and Methods: An analytical cross-sectional study was done in a tertiary care center in South India for 1 year. Sonographically confirmed NAFLD patients of the age group 18 years and above were consecutively enrolled for the study after getting informed consent. Anthropometric measurements, ultrasonography for identifying the grades of fatty liver and FDG-PET were performed in the study participants. The data for the study were collected by the research personnel and entered in Microsoft Excel. The data were analyzed in the IBM SPSS version 20.0 software. Results: A total of 24 patients were in the final analysis. The mean age of patients in this study was 56.79 (11.26) years. Among the 24 patients, 95.83% (n = 23) were males. The mean FDG-PET uptake in the carotids was 1.75 (0.42) units. The FDG uptake was higher in the moderate NAFLD group (1.46 [0.40] v/s 2.04 [0.14]) and the difference was statistically significant with P < 0.001. The FDG uptake between the coronary artery disease (CAD) with NAFLD and non CAD with NAFLD groups was not statistically significant (1.60 [0.46] v/s 1.86 [0.36], P = 0.17). The FDG uptake in CAD patients with mild and moderate NAFLD had no statistical significance between the two groups (1.43 [0.45] v/s 2.00 [0.00], P= 0.06). Conclusion: The findings of this study showed increased uptake of FDG-PET in carotids in subjects with moderate fatty liver when compared with those with mild fatty liver.

Fungal Colonization and Infections-Interactions with Other Human Diseases.

Candida albicans is a commensal fungus that asymptomatically colonizes the skin and mucosa of 60% of healthy individuals. Breaches in the cutaneous and mucosal barriers trigger candidiasis that ranges from asymptomatic candidemia and mucosal infections to fulminant sepsis with 70% mortality rates. Fungi influence at least several diseases, in part by mechanisms such as the production of pro-carcinogenic agents, molecular mimicking, and triggering of the inflammation cascade. These processes impact the interactions among human pathogenic and resident fungi, the bacteriome in various organs/tissues, and the host immune system, dictating the outcomes of invasive infections, metabolic diseases, and cancer. Although mechanistic investigations are at stages of infancy, recent studies have advanced our understanding of host-fungal interactions, their role in immune homeostasis, and their associated pathologies. This review summarizes the role of C. albicans and other opportunistic fungi, specifically their association with various diseases, providing a glimpse at the recent developments and our current knowledge in the context of inflammatory-bowel disease (IBD), cancers, and COVID-19. Two of the most common human diseases where fungal interactions have been previously well-studied are cancer and IBD. Here we also discuss the emerging role of fungi in the ongoing and evolving pandemic of COVID-19, as it is relevant to current health affairs.

Recognition and Chaperoning by Pex19, Followed by Trafficking and Membrane Insertion of the Peroxisome Proliferation Protein, Pex11.

Pex11, an abundant peroxisomal membrane protein (PMP), is required for division of peroxisomes and is robustly imported to peroxisomal membranes. We present a comprehensive analysis of how the Pichia pastoris Pex11 is recognized and chaperoned by Pex19, targeted to peroxisome membranes and inserted therein. We demonstrate that Pex11 contains one Pex19-binding site (Pex19-BS) that is required for Pex11 insertion into peroxisomal membranes by Pex19, but is non-essential for peroxisomal trafficking. We provide extensive mutational analyses regarding the recognition of Pex19-BS in Pex11 by Pex19. Pex11 also has a second, Pex19-independent membrane peroxisome-targeting signal (mPTS) that is preserved among Pex11-family proteins and anchors the human HsPex11γ to the outer leaflet of the peroxisomal membrane. Thus, unlike most PMPs, Pex11 can use two mechanisms of transport to peroxisomes, where only one of them depends on its direct interaction with Pex19, but the other does not. However, Pex19 is necessary for membrane insertion of Pex11. We show that Pex11 can self-interact, using both homo- and/or heterotypic interactions involving its N-terminal helical domains. We demonstrate that Pex19 acts as a chaperone by interacting with the Pex19-BS in Pex11, thereby protecting Pex11 from spontaneous oligomerization that would otherwise cause its aggregation and subsequent degradation.

Balancing the Opposing Principles That Govern Peroxisome Homeostasis.

Despite major advances in our understanding of players and mechanisms involved in peroxisome biogenesis and peroxisome degradation, very few studies have focused on unraveling the multi-layered connections between, and the coordination of, these two opposing processes that regulate peroxisome homeostasis. The intersection between these processes also provides exciting avenues for future research. This review highlights the links between peroxisome biogenesis and degradation, incorporating an integrative approach that is critical not only for a mechanistic understanding, but also for manipulating the balance between these processes in relevant disease models.

Immuno-MALDI MS dataset for improved detection of HCVcoreAg in sera.

Complicated and large-scale challenge the contemporary biomedical community faces are development of highly-sensitive analytical methods for detection of protein markers associated with development of pathogenic mechanisms [2]. The atomic force microscopy (AFM) method in combination with specific fishing is unique among other analytical protein detection approaches; it allows visualization and counting of single protein molecules [3-6]. The present dataset focus on mass spectrometry method for detection of human hepatitis C virus core antigen (HCV core Ag) taking into account the potential modification with cations in blood serum samples, using mica chips for the atomic force microscopy (AFM-chips). To conduct specific protein fishing, we used flat AFM-chips preliminary sensibilized with molecular probes - aptamers, which are single-stranded DNA sequences. In our study we used four types of aptamers up to 85 nucleotides specific against the target protein - HCVcoreAg [3,4]. Working (n = 19) and control (n = 11) AFM-chips with aptamers were preliminarily immobilized on the surface in four zones and incubated in blood serum samples (See Supplementary fig. 1). Analysis of MS data regarding modification of marker protein peptides with Na+, K+, K2Cl+, and Na2Cl + ions enables to enhance the reliability of target proteins detection in the serum thereby demonstrating a high diagnostic potential.

Age and Body Mass Index Has No Adverse Effect on Clinical Outcome of Unicompartmental Knee Replacement - Midterm Followup Study.

INTRODUCTION: Unicompartmental knee replacement (UKR) is well-established procedure for the anteromedial compartment of knee arthritis with intact anterior cruciate ligament. The significance of age and body mass index (BMI) is not clear in the outcomes of UKR. Our hypothesis was that age and BMI does not affect the clinical and functional outcome following fixed bearing UKR. MATERIALS AND METHODS: The study cohort of 148 was selected after stringent inclusion criteria and average followup was 5.6 years (range 2-10 years). The fixed bearing Miller Galante UKR procedure was carried out on all patients. RESULTS: In the study cohort of 175, the average age of the cohort was 61.7 years. The sample size aged ≤55 years and aged ≥55 years was 38 and 137, respectively. The mean BMI of the cohort was 29.2 kg/m2 (range: 21-38 kg/m2). The sample size of BMI ≤30 kg/m2 and BMI ≥30 kg/m2 was 117 and 58, respectively. In the cohort group, BMI ≤30 kg/m2 and BMI ≥30 kg/m2, there was no statistically significant difference in the Knee Society Score clinical scores, functional scores, and knee range of motion scores, (P > 0.05). This study infers no statistically significant difference in the clinical and functional outcome between age group ≤55 years and age ≥55 years, (P > 0.05). The failure rates of the group of BMI ≤30 kg/m2 and BMI ≥30 kg/m2 were 4.27% (5 knees) 3.44% (2 knees), respectively. The failure rates in the age group ≤55 years and group ≥55 years were 2 knees (3.44%) and 5 knees (4.27%), respectively. CONCLUSION: This study confirms that age and BMI does not influence the functional outcome and clinical outcome following fixed bearing UKR.

Peroxisome biogenesis, membrane contact sites, and quality control.

Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.

18F-FDG PET/CT in the evaluation of cancer cervix: Where do we stand today?

The incidence of gynecological malignancies is on the rise partly because of the availability of screening programmes, awareness, higher technological advancements, and availability of better medical care. Early diagnosis of any malignancy leads to prompt treatment. Use of 18Fluorine-Fluorodeoxyglucose (F-FDG) PET/CT in the treatment and follow-up of patients with Ca cervix considerably improves patient management. The primary diagnosis of Ca cervix is made either by biopsy of a visible tumor on the cervix or by a cone biopsy of a nonvisible malignant cervical focus. The staging procedure is purely clinical (i.e. gynecologic examination under general anesthesia) according to the International Federation of Gynaecology and Obstetrics classification. Earlier, with the nonavailability of sophisticated medical equipment and imaging specialists, oncologists relied heavily on clinical examination. However, anatomical and functional imaging has been proven to be considerably superior in understanding parametrial involvement and nodal/distant metastases in the cancer cervix than clinical examination alone. Data are evolving on the usage of F-FDG PET/CT in initial staging, treatment planning, and monitoring therapy response for gynecological malignancies. Prognostic information derived from the primary lesion such as the maximum standardized uptake value, metabolic tumor volume, and extent of para-aortic nodal metastatic disease plays a critical role in tailoring therapy on the basis of patient tumor-specific factors rather than on International Federation of Gynaecology and Obstetrics stage alone. Thus, F-FDG PET/CT needs to be listed not only under the panel of pretherapy investigations for Ca cervix but also for recurrence and therapy response assessments. It allows a more confident approach to patient management at initial staging, especially in terms of the decision to choose surgical versus palliation measures.

Unusual Sites of Metastatic and Benign I 131 Uptake in Patients with Differentiated Thyroid Carcinoma.

INTRODUCTION: Differentiated thyroid carcinoma (DTC) is the most common pathological type of thyroid carcinoma, which includes papillary and follicular subtypes. DTC is usually indolent, characterized by good prognosis, and long-term survival. Total thyroidectomy is the mainstay of treatment in DTC which is followed by diagnostic whole body 131I (WBI) scan. Like other primary malignancies of the head and neck, DTC follows a consistent pattern of spread in the cervical LNs. The central compartment, level VI and VII, is the first sentinel node followed by spread to the lateral compartments levels II-V, followed by the contralateral side. Inspite of nodal involvement, DTC usually have a favourable outcome. Presence of extrapulmonary distant metastases could predict a poor prognosis for high-dose 131I therapy. However, distant metastasis occurs often as a grave event and mortality rates vary depending on metastatic sites. AIM AND OBJECTIVES: A range of rare 131I concentrating DTC deposits in sella, orbit, choroid, skeletal muscles, liver, skin, costochondral soft tissue, pancreas and kidney, and a few benign 131I concentrating sites are being depicted. MATERIALS AND METHODS: Metastatic sites from DTC can be easily identified by performing a whole body 131I (WBI) scan along with a stimulated thyroglobulin (Tg) estimation (TSH >30 uIU/ml). Apart from thyroid and thyroid-related diseases, certain benign non-thyroidal pathologies can concentrate radioiodine (131I). From 13,000 of our patients who underwent radioiodine scan for thyroid cancer, we have selected a few cases of 131I concentrating benign and malignant lesions for illustration. RESULTS: Out of 13000 DTC patients who underwent whole body 131I scintigraphy in our department from Jan 2007 till Mar 2018, 25 patients revealed benign sites of 131I uptake. 61 % patients had residual thyroid tissue with or without associated nodal involvement. Remaining patients had distant metastases. Rare sites of functioning thyroid metastases and benign sites of I 131 uptake have been selected for illustration. CONCLUSION: Apart from the WBI (two-dimensional, planar) images, single-photon emission computed tomography-computed tomography (SPECT-CT) has been incremental in localizing benign lesions which greatly depends on their location. This pictorial review highlights the need to create an awareness to detect metastatic deposits of DTC at unexpected sites. Otherwise patients will need further investigation to rule out unsuspected sites of functioning distant metastases.

Medium term results of Avon patellofemoral joint replacement.

BACKGROUND: Ten to fifteen percent of knee arthritis is reported to be isolated patellofemoral arthritis. Total knee arthroplasty is not recommended for isolated patella femoral arthritis particularly in young patients. We present the retrospective review of 45 consecutive patellofemoral replacements performed in 41 such patients, between June 2002 and January 2007. MATERIALS AND METHODS: All patients were operated by single surgeon (SM) or under his supervision. All forty five patients had minimum three year followup and had the data collected prospectively. No patient was lost to followup. This data was later collated by review of notes, radiographs, and a clinical followup. The patients were assessed using knee function score and Melbourne patellofemoral score. RESULTS: The average followup was 4.5 years. The preoperative average Melbourne (Bartlett) score was 10 (range 5-21). Preoperative knee functional score averaged 57 (range 23-95). The average range of movement was 116° (range 100°-140°). Postoperatively, the average Melbourne knee score improved to 25 (range 11-30), while the knee function score was 85 (range 28 - 100). The difference was statistically significant (P<0.05). Eighty-five percent rated the result as good or excellent, while 12% rated it as fair. Five percent thought the result was poor. The most common complaint was clicking at 40° of flexion (n=7). Six patients underwent arthroscopic lateral release, which improved the symptoms in four patients. Two knees were revised one due to progression of tibiofemoral arthritis and the other due to persistent clicking, yielding a survival rate of 95.6% at an average five year followup. CONCLUSION: The Avon patellofemoral joint replacement provides predictably good results and excellent survivorship in the medium term, for isolated patellofemoral arthritis. However, progression of tibiofemoral arthritis remains unpredictable and therefore patient selection is crucial to ensure success. Clicking remains a potential problem and can compromise the postoperative results in upto 15% of the cases.

3-Cyclopropyl-1,2,4-triazolo[3,4-b]benzothiazole monohydrate.

The title structure, C11H9N3S.H2O, contains two crystallographically independent molecules, each consisting of a 1,2,4-triazolo[3,4-b]benzothiazole-fused ring fragment substituted with a cyclopropyl ring, and a water molecule. The geometry of both molecules differs slightly. Both molecules are planar within 0.041 and 0.03 A. The dihedral angle between triazole and the cyclopropyl ring is 60.0(1) and 61.7(1) degrees for molecules A and B, respectively, and the cyclopropyl ring is inclined at an angle of 2.0(1) and 1.2(1) degrees to the benzothiazole moiety for molecules A and B, respectively. The packing of the molecules is stabilized by O-H...N-type hydrogen bonds.