A Protease from Moringa oleifera Lam. Exhibits In-vitro Blood Clot Solubilization and Fibrin Hydrolysis.

Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. A fibrinolytic protease was isolated from fresh leaves of Moringa oleifera Lam. and characterized for its potential to solubilize blood clots and hydrolyse fibrin under in-vitro conditions. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 oC with 50 µg protein. The fibrinolytic activity of isolated protease was also confirmed by fibrin zymography. Km and Vmax of isolated protease were determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clots by 96 h under in-vitro conditions. In-vitro fibrin hydrolysis and blood clot solubilization activities shown by an isolated protease from leaves of Moringa oleifera Lam. suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis.

Pseudomonas aeruginosa PR23 isolated from oil contaminated soil tolerate and degrades mixture of polyaromatic hydrocarbons and express novel proteins.

Pseudomonas aeruginosa PR23 isolated from the hydrocarbon contaminated soil can tolerate and degrade mixture of polyaromatic hydrocarbons (PAHs) at an initial concentration of 1300 ppm. The degradation and intermediates formed were assessed by gas chromatography-mass spectrometry (GC-MS) analysis. The isolated strain was able to degrade 59.2% of the mixture of PAHs in 3 days and 71.6% by day 15. Effect of PAHs on protein expression in Pseudomonas aeruginosa PR23 was studied using nano LC-MS/MS. Thirty-six proteins showed a more than 2-fold increase in expression in the presence of mixture of PAHs. Out of these proteins, 7 proteins have been reported for their role in degradation of naphthalene, phenanthrene, and pyrene. The data revealed the presence of 16 proteins that were uniquely expressed in the presence of mixture of PAHs. A twin-arginine translocation signal peptide (Tat system), known for the transportation of folded proteins across the cell membrane, showed more than 8-fold increased expression in the presence of mixture of PAHs. These results indicate that the isolated strain adopts the conditions in the presence of mixture of PAHs by modulating its metabolic and physiological processes. These findings suggest that Pseudomonas aeruginosa PR23 may be a suitable candidate for use in the development of strategies for bioremediation of mixtures of PAHs.

Comparative evaluation of efficacy and safety of calcipotriol versus calcitriol ointment, both in combination with narrow-band ultraviolet B phototherapy in the treatment of stable plaque psoriasis.

BACKGROUND: Vitamin D analogues and NBUVB are both well-recognised modes of therapy in the treatment of chronic stable plaque psoriasis. The objective of this open label intraindividual, left right study was to compare two different vitamin D analogues, calcipotriol and calcitriol, in combination with NBUVB phototherapy in psoriasis. METHODS: Thirty patients with stable plaque psoriasis were enrolled for a 12-week clinical trial. The target lesion on the left side was treated topically with calcitriol ointment, while that on the right side was treated with calcipotriol ointment once daily. The whole body was irradiated with narrow-band ultraviolet B phototherapy (NBUVB) three times per week. Efficacy was assessed by target plaque scoring. RESULTS: Both therapies resulted in a statistically significant reduction in erythema, scaling, thickness, and target plaque score, seen as early as 2 weeks into therapy. However, the calcipotriol combination led to an earlier clearance of plaques and a lesser relapse rate than the calcitriol combination. The number of treatment sessions and cumulative NBUVB doses were significantly lower in the calcipotriol-treated group. CONCLUSION: Both vitamin D analogues appear to be safe, effective, and cosmetically acceptable, with calcipotriol being more efficacious, well tolerated, with a rapid onset of action and a better maintenance of response.

A Non-competitive Serpin-Like Thrombin Inhibitor Isolated from Moringa oleifera Exhibit a High Affinity for Thrombin.

The majority of the clotting factors involved in blood coagulation pathways are serine proteases and thrombin is one of the key serine proteases involved in blood clotting. Many synthetic and chemical drugs targeting these proteases as therapeutics are known. However, they are associated with serious side effects such as bleeding, haemorrhage, edema etc. Serine protease inhibitors from plants have been suggested as one of the potential anticoagulant molecules against thrombosis. In the present work, a direct thrombin inhibitor from Moringa oleifera was isolated, purified and characterized. The homogeneity of the inhibitor is confirmed on native- PAGE. The purified inhibitor (5 µg) showed 63% thrombin inhibition at pH 7.2 at 37 °C. The IC50 value of the isolated inhibitor was determined as 4.23 µg. The inhibitor on SDS-PAGE appeared as a single protein-stained band corresponding to 50 kDa thereby indicating its molecular weight as 50 kDa. Purified thrombin inhibitor (5 µg) showed 12% inhibition of trypsin, and 17% inhibition of chymotrypsin. This suggests more specificity of purified inhibitor towards thrombin. The isolated inhibitor showed a non-competitive mode of inhibition against thrombin as determined by the Dixon plot. The inhibition constant (Ki) was calculated as 4.35 × 10-7 M. The present work reports for the first time a direct thrombin inhibitor from M. oleifera which may be further explored as an antithrombotic drug.

A cystatin C similar protein from Musa acuminata that inhibits cathepsin B involved in rheumatoid arthritis using in silico approach and in vitro cathepsin B inhibition by protein extract.

Rheumatoid arthritis (RA) is an auto-immune disease that affects the synovial lining of the joints, causes synovitis and culminates to joint destruction. Cathepsin B is responsible for digesting unwanted proteins in extracellular matrix but its hyper expression could implicate in pathological diseases like RA. Available treatments for RA are classified into non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), and steroids, but the severe side effects associated with these drugs is one of concerns and cannot be ignored. Thus, any alternative therapy with minimum or no side effects would be a cornerstone. In our in silico studies a cystatin C similar protein (CCSP) has been identified from Musa acuminata that could effectively inhibit the cathepsin B activity. In silico and molecular dynamics studies showed that the identified CCSP and cathepsin B complex has binding energy -66.89 kcal/mol as compared to cystatin C - cathepsin B complex with binding energy of -23.38 kcal/mol. These results indicate that CCSP from Musa acuminata has better affinity towards cathepsin B as compared to its natural inhibitor cystatin C. Hence, CCSP may be suggested as an alternative therapeutic in combating RA by inhibiting its one of the key proteases cathepsin B. Further, in vitro experiments with fractionated protein extracts from Musa sp. peel inhibited cathepsin B to 98.30% at 300 µg protein concentration and its IC50 was found to be 45.92 µg indicating the presence of cathepsin B inhibitor(s) in protein extract of peel which was further confirmed by reverse zymography.Communicated by Ramaswamy H. Sarma.

Identification of thrombin inhibiting antithrombin-III like protein from Punica granatum using in silico approach and in vitro validation of thrombin inhibition activity in crude protein.

Thrombosis is characterized by the formation of clots in the blood vessels. Antithrombin-III deficiency in the blood causes thrombus formation. Supplementing antithrombin-III may serve as anticoagulant therapy. In the present studies, an antithrombin like Protein from Punica granatum has been identified and characterized using in silico approach. Based on sequence homology, an ALPP was selected depending upon its highest binding affinity of -41.28 kcal/mol with thrombin. Thrombin structure complexed with ALPP was docked with TAME using AutoDock Vina. No binding was observed for TAME at Ser195 of thrombin. MD simulation (50 ns) was performed to evaluate the flexibility and stability of docked complexes. In vitro assays with crude protein showed 78% thrombin inhibition at 5 µg and calculated IC50 value was 0.188 µg. The presence of thrombin inhibitors in crude protein was also confirmed by reverse zymography. Thus, it is very likely that the protein identified from P. granatum may act as thrombin inhibitor.

Scrutinising the role of simvastatin in a patient of Pachyonychia Congenita with KRT6A gene mutation.

A 25-year-old male patient presented with palmoplantar keratoderma, dystrophic nails, severe plantar pain and oral leukokeratosis since birth. On genetic analysis, a heterozygous KRT6A gene missense mutation (c.1381G > A, p.Glu461Lys in exon 7) was identified by next-generation sequencing technology, consistent with pachyonychia congenita 6a. Oral simvastatin 40 mg was started once daily, and after 16 weeks of therapy, excellent improvement was noted in palmoplantar keratoderma and plantar pain. The maximum thickness of his foot callosity reduced by 4 mm on ultrasonography, and the Dermatology Life Quality Index score dropped significantly by eight points. These benefits may be attributed to inhibition of KRT6A gene expression, modulation of autophagy and mitophagy and Keap1-Nrf2 signalling activation; the latter two mechanisms of statins previously undiscussed in the context of pachyonychia congenita. Simvastatin and other statins are pathogenesis-targeted, disease-modifying therapy in pachyonychia congenita, therefore qualifying as a promising treatment avenue and warranting further clinical trials.

Is NAT2 Gene Polymorphism Associated with Vitiligo?

BACKGROUND: N-acetyltransferase-2 (NAT2) is a phase II xenobiotic enzyme that plays an important role against oxidative stress-mediated reactive oxygen species protection. Polymorphism in specific genotypes of NAT2 may lead to increase an imbalance in antioxidant systems and may influence the pathogenesis of vitiligo. We conducted this study to see the association between NAT2 gene polymorphism and risk of vitiligo. We looked into whether single-nucleotide polymorphisms (SNP) at positions 857, 481 and 590 of the coding region of the NAT2 gene play as a risk factor for vitiligo among north Indian people. METHODS: In this study, we assessed 100 patients with vitiligo and 160 healthy individuals as controls. Genomic DNA was extracted from human peripheral blood and polymerase chain reaction-restricted fragment length polymorphism was done to identify the single nucleotide polymorphism at positions 857, 481, and 590 of the coding region of the NAT2 gene. RESULTS: In this study, we observed a significant higher risk with slow acetylator genotypes of NAT2 (OR = 2.85; 95% CI = 1.68-4.84, P value < 0.001) for the vitiligo. Furthermore, in the association between NAT2 acetylator genotypes with percentage of body surface area (BSA) of disease, we observed that slow acetylator genotypes of NAT2 has significant higher risk with low grade of disease (1%-10% >11%-30% >30% of BSA). LIMITATIONS: A major limitation of this study was the small sample size and warrants further investigation on a large epidemiological study to confirm these findings. CONCLUSIONS: Our preliminary data indicate that NAT2 slow acetylator genotype exhibits significant association for the risk of vitiligo, especially in disease predisposition and initiation.

Literature review of oral tuberculosis and report of a case with unique histological presentation.

As a consequence of the emergence of drug resistant tuberculosis (TB) and various immuno-compromised states, there is a re-emergence of many forgotten extrapulmonary manifestations of TB including oral TB, which must be taken into consideration while diagnosing oral lesions. The present article discusses the geographical burden, temporal evolution, demographic variables, clinical presentation and treatment of oral TB. The occurrence is most commonly secondary to pulmonary TB but oral symptoms may precede systemic symptoms. The most common presentation is ulceration (71%) and histopathological specimens demonstrate the characteristic epithelioid and langhans cells. In a unique case, presented here, an ulcerative tuberculous gingival lesion demonstrated dense plasma cell infiltration histologically and closely mimicked plasma cell gingivitis which made the diagnosis challenging.

Effect of periodontal disease on electromyographic activity of muscles of mastication: A cross-sectional study.

AIM: This study aims to compare surface electromyographic activities (EMG) of four muscles, that is anterior temporalis (AT), masseter (MST), sternocleidomastoid (SCM) and anterior belly of digastric (ABD), between chronic periodontitis patients and periodontally healthy individuals as well as to correlate these EMG findings with periodontal parameters. METHODS: Thirty chronic periodontitis patients were recruited in Group I and 30 periodontally healthy individuals in Group II. Clenching and resting EMG for 4 muscles (AT, MST, SCM and ABD) were recorded for each participant. These EMG values were compared between the groups and correlated with periodontal parameters (plaque index, modified sulcular bleeding index, probing depth and clinical attachment loss). RESULTS: Resting EMG (rEMG) showed no significant difference between the groups for any muscle. Clenching EMG (cEMG) values were significantly lower in Group I than Group II for AT and MST (P = .001), but not so for SCM and ABD. Within Group I, AT and MST showed a significant negative correlation of cEMG with probing depth and clinical attachment loss (P < .05). CONCLUSION: Electrical muscle activities of AT and MST are lower in chronic periodontitis than periodontally healthy individuals.

Acquired syphilis in children: A retrospective study over two-and-a-half decades in a tertiary care center in northern India.

BACKGROUND: Syphilis causes considerable morbidity not only in adults but also in children. Unfortunately, acquired syphilis in children has not received due attention and remains a relatively ignored field. METHODS: This was a retrospective study, in which epidemiologic, clinical, and investigational data were analyzed from medical records of all sexually transmitted infection (STI) cases attending the STI clinic of Pt. B. D. Sharma, PGIMS, Rohtak from January 1992 to November 2018. Children of age ≤14 years with acquired syphilis were studied. RESULTS: Of 9138 STD cases registered from January 1992 to November 2018, 39 were children with acquired syphilis, of whom 22 were males and 17 were females. The majority were in 12-14 years age group (22 cases; 56.41%). A history of sexual abuse could be elicited in 10 patients. Three gave a history of voluntary sexual activity. Examination revealed secondary syphilis in 32, latent syphilis in 6 and only one case of primary chancre. Analysis of incidence of pediatric acquired syphilis cases over three-year intervals during the last 27 years indicated an initial decreasing trend followed by recent slight rise in last 3 years. CONCLUSION: Social inhibitions and reluctance to report child sexual abuse may have led to an underestimate of actual prevalence. The prevalence in the 12- to 14-year age group may indicate emerging trends of promiscuous sexual behavior. The recent rising trend in occurrence of childhood acquired syphilis may mirror the recent resurgence of adult syphilis.

Effectiveness of anterior middle superior alveolar injection using a computer-controlled local anesthetic delivery system for maxillary periodontal flap surgery.

BACKGROUND: Profound anesthesia with adequate duration is required in periodontal flap surgery, which involves the manipulation of both hard and soft tissues. The anterior middle superior alveolar (AMSA) injection may be an alternative to multiple injections required for this purpose in the maxilla. The present study aimed to assess the effectiveness of AMSA injection using computer-controlled local anesthetic delivery (CCLAD) system to anesthetize buccal hard tissue (BHT), buccal soft tissue (BST), palatal hard tissue (PHT), and palatal soft tissue (PST) around the maxillary teeth. METHODS: Thirty-five patients who were indicated for open flap debridement in a whole maxillary quadrant were given AMSA injection using the CCLAD. The effectiveness of anesthesia was evaluated using subjective and objective parameters around each tooth. Supraperiosteal infiltrations were administered to complete the surgery wherever the AMSA injection was ineffective. RESULTS: The AMSA injection was more effective on the palatal tissues than on the buccal tissues, as 94.14% of PST and 87.89% of PHT sites were anesthetized compared to 49.22% and 43.75% of BHT and BST sites, respectively. There was no significant difference in the frequency of anesthesia around the anterior and posterior teeth. The PHT was significantly more anesthetized (P = 0.003) in males than in females. CONCLUSIONS: The AMSA injection using CCLAD is highly effective on palatal tissues and could be used as a first-line anesthesia for periodontal flap surgery. However, its effect on buccal tissues is less predictable, with supraperiosteal infiltration often required to supplement the AMSA injection.

Comparative evaluation of bioburden and sterility of indigenously prepared bone allograft with and without gentamicin.

During bone allograft processing, despite stringent donor screening and use of aseptic techniques, microbial invasion may occur due to the porous nature of the graft and cause potentially fatal infections. The aim of the present study was to prepare bone allograft with and without gentamicin and to compare bioburden and sterility in the obtained grafts to evaluate the role of antibiotic in enhancing graft safety. Fifty samples of demineralized freeze-dried bone allograft were prepared from suitable donors according to international standards. Randomly selected 25 samples were placed in 8 mg gentamicin/gram bone solution for 1 h. Packaging and sealing was done to ensure no microbial ingress during transportation. 40 samples were selected for bioburden testing. Remaining 10 were subjected to 25 kGy gamma radiation and tested for sterility. Microbiological evaluation revealed no evidence of colony forming units in all the samples of both the groups (Bioburden = 0). Post-radiation sterility testing also revealed no bacterial colony in the tested samples from both the groups. Favorable results validate the processing protocol while comparable results in both groups indicate no additive benefit of gentamicin addition. Nil bioburden may be used in further studies to determine a lower radiation dose to achieve adequate sterility and minimize the disadvantages of radiation like collagen cross-linking and decreased osteoinductive capacity.

Extensive condylomata lata in an adolescent: An uncommon and unusual presentation.

Sexually transmitted infections (STIs) in children account for a great physical and psychosocial morbidity. Being rare, they require a high index of suspicion for diagnosis. STIs may also be the first indication of child sexual abuse (CSA). Consequences of an undetected infection are potentially devastating; conversely, a false suspicion can have equally detrimental effects on both the child and the parents. Herein, we report a case of acquired secondary syphilis in a child presenting with unusually extensive condylomata lata at multiple sites and review the literatue to understand the possible interrelationship of syphilis and CSA.