Screening for antibacterial and cytotoxic activities of Sri Lankan marine sponges through microfractionation: Isolation of bromopyrrole alkaloids from Stylissa massa.

Sri Lanka is a biodiversity hotspot and one of the richest geographical locations of marine sponges in the Indian ocean. However, the most extensive taxonomical study on Sri Lankan sponge biodiversity dates back ~100 years and only a limited number of studies have been conducted on sponge natural products. In the current study, 35 marine sponge specimens (collected from 16 sponge habitats around Sri Lanka) were identified, microfractionated and evaluated for antibacterial and anticancer assays. In total, 30 species were characterized, of which 19 species gave extracts with antibacterial and/or cytotoxic activities. Microfractionated organic extract of Aciculites orientalis gave the most potent antibacterial activity against Staphylococcus aureus and strongest lymphoma cell toxicity was exhibited by the organic extract of Acanthella sp. Guided by the molecular ion peaks in the bioactive fractions, large-scale extraction of Stylissa massa led to the isolation of three bromopyrrole alkaloids, sceptrin, hymenin and manzacidin A/C. Of these, sceptrin exhibited broad spectrum antibacterial activity against both Escherichia coli and S. aureus (MIC of 62.5 µM against both species). Based on natural product literature, seven promising species were identified as understudied. Their further exploration may lead to the discovery of structurally novel compounds.

Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS-CoV-2 activity.

The recent COVID-19 pandemic shows the critical need for novel broad spectrum antiviral agents. Scorpion venoms are known to contain highly bioactive peptides, several of which have demonstrated strong antiviral activity against a range of viruses. We have generated the first annotated reference transcriptome for the Androctonus amoreuxi venom gland and used high performance liquid chromatography, transcriptome mining, circular dichroism and mass spectrometric analysis to purify and characterize twelve previously undescribed venom peptides. Selected peptides were tested for binding to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and inhibition of the spike RBD - human angiotensin-converting enzyme 2 (hACE2) interaction using surface plasmon resonance-based assays. Seven peptides showed dose-dependent inhibitory effects, albeit with IC50 in the high micromolar range (117-1202 µM). The most active peptide was synthesized using solid phase peptide synthesis and tested for its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7). On exposure to the synthetic peptide of a human lung cell line infected with replication-competent SARS-CoV-2, we observed an IC50 of 200 nM, which was nearly 600-fold lower than that observed in the RBD - hACE2 binding inhibition assay. Our results show that scorpion venom peptides can inhibit the SARS-CoV-2 replication although unlikely through inhibition of spike RBD - hACE2 interaction as the primary mode of action. Scorpion venom peptides represent excellent scaffolds for design of novel anti-SARS-CoV-2 constrained peptides. Future studies should fully explore their antiviral mode of action as well as the structural dynamics of inhibition of target virus-host interactions.

Epitopes Displayed in a Cyclic Peptide Scaffold Bind SARS-COV-2 Antibodies.

The SARS-CoV-2 virus that causes COVID-19 is a global health issue. The spread of the virus has resulted in seven million deaths to date. The emergence of new viral strains highlights the importance of continuous surveillance of the SARS-CoV-2 virus by using timely and accurate diagnostic tools. Here, we used a stable cyclic peptide scaffolds to present antigenic sequences derived from the spike protein that are reactive to SARS-CoV-2 antibodies. Using peptide sequences from different domains of SARS-CoV-2 spike proteins, we grafted epitopes on the peptide scaffold sunflower trypsin inhibitor 1 (SFTI-1). These scaffold peptides were then used to develop an ELISA to detect SARS-CoV-2 antibodies in serum. We show that displaying epitopes on the scaffold improves reactivity overall. One of the scaffold peptides (S2_1146-1161_c) has reactivity equal to that of commercial assays, and shows diagnostic potential.

In Vitro Biomedical and Photo-Catalytic Application of Bio-Inspired Zingiber officinale Mediated Silver Nanoparticles.

To minimize the hazardous effect of physical and chemical synthesis of nanoparticles we focused on the green synthesis of nanoparticles. Nanotechnology is a research hotspot and catch great attention because of its versatile applications in medical, biosciences and engineering fields. Purpose of our recent study is to synthesize bio-inspired metallic silver NPs by root mediated Zingiber officianale extract. The synthesized Ag-NPs were further characterized by using UVVisible spectroscopy, XRD, EDX, SEM, TEM and DLS techniques. The extent of crystallites were confirmed by X-ray diffraction. SEM and TEM revealed the morphological features with size of nanoparticles between 17.3 and 41.2 nm. FTIR analysis confirmed the capping of nanoparticles by bio active constituents present in Zingiber officinale extract. Later EDX confirmed the elemental composition of nanoparticles. Zeta potential, PDI and hydrodynamic size of Ag-NPs were confirmed by DLS. The synthesize Ag-NPs possess eminent biological potency against bacterial and leishmanial strains. Moreover considerable anti-diabetic, anticancer, antioxidant and biocompatibility nature of Ag-NPs was elucidated. The highest antioxidant activity of 50.61± 1.12%, 38.22 ± 1.18% and 27.39 ± 0.92 at 200 g/mL for TAC, TRP DPPH and was observed respectively. Ag-NPs exhibit potent leishmanicidal activity of 80% ± 1.4 against promastigotes and 77% ± 1.6 against amastigotes cultures of L. tropica. Highest antidiabetic activity 30 ± 0.77% recorded at 200 µg/ml. Highest Brine shrimps cytotoxicity of Ag-NPs was 60 ± 1.18 at 200 g/ml. Maximum dye degradation for Ag-NPs was recorded as 94.1% at 140 minute. All UTI isolates were resistant to antibiotics not coated with Ag-NPs. By applying 1% of Ag-NPs highest activity was recorded as 25 ± 1.58 mm against K. pneumoniae. Maximum zone of inhibition for Ag-NPs coated with Imipenem antibiotics 26 ± 1.5 mm against K. pneumoniae and coated with Ciprofloxacin 26 ± 1.4 m against S. aureus were measured. Last but not least high biocompatible nature of Ag-NPs was observed against fresh RBCs making the ecofriendly biosynthesized silver NPs a multi-dimensional candidate in biomedical field.

Screening for bioactive secondary metabolites in Sri Lankan medicinal plants by microfractionation and targeted isolation of antimicrobial flavonoids from Derris scandens.

ETHNOPHARMACOLOGICAL RELEVANCE: Sri Lanka is known to have very diverse flora. Many of these species are used for plant-based remedies, which form the integral part of two Sri Lankan systems of traditional medicine, Ayurveda and Deshiya Chikitsa. Despite their widespread use, only a limited number of studies have probed into the scientific evidence for bioactivity of these medicinal plants. Such studies rarely progress to the identification of bioactive natural products. AIM OF THE STUDY: The primary aim was to develop a bioactivity screening method and apply it to 50 Sri Lankan medicinal plants where antimicrobial properties could be relevant for its traditional use. The subsequent aim was the progression into defining and characterising potent isolates within targeted compound classes from such plants, i.e. Derris scandens and its antimicrobial flavonoids. MATERIAL AND METHODS: The plant collection comprised 24 species of Fabaceae, 15 Rubiaceae, 7 Solanaceae and 4 Cucurbitaceae plants. These 50 species were collected based on their ethnopharmacological importance and use in Sri Lankan traditional medicine. Crude extracts from each species were initially subjected to radial disc diffusion and microdilution assays. Subsequently, aqueous extracts of all plants were microfractionated in deep well plates using reversed-phase HPLC. Fractions were tested for antibacterial and cytotoxic activities and masses of target bioactive compounds were identified using mass spectrometry. Bioactive compounds with the masses identified through microfractions were isolated from Derris scandens using reversed-phase HPLC. The isolated pure compounds were characterised using LC-MS and NMR. RESULTS: Crude aqueous extracts from 19 species showed activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) in the radial disc diffusion assay. Crude aqueous extracts from 34 plant species and organic extracts from 46 plant species were active against S. aureus (≤4 mg mL-1) in the microdilution assay. Microfractionation demonstrated antibacterial activity for 19 plants and cytotoxicity for 6 plants. Furthermore, target bioactive compounds and their molecular ions were identified during microfractionation. Dalpanitin and vicenin-3, two of the flavonoids isolated from Derris scandens gave MICs of 23 µg mL-1 against S. aureus. Dalpanitin also exhibited relevant MICs on Gram-negative bacteria (94 µg mL-1 against Escherichia coli and Pseudomonas aeruginosa). CONCLUSION: The microfractionation protocol developed in this study enabled time-efficient screening of many plants species, using a small quantity of sample material. In addition, microfractionation served as a guiding tool for identifying individual antimicrobial compounds. Through this process, flavonoids were isolated from Derris scandens, out of which dalpanitin and vicenin-3 showed activity in the low micromolar range. The high hit rate for in vitro antibacterial properties from this ethnopharmacologically guided sample collection gives credence to Sri Lankan traditional herbal medicine as a source for drug discovery.

Cotton productivity enhanced through transplanting and early sowing

Due to high temperatures in arid regions, cotton crop emergence and early establishment of seedlings are adversely affected. Field studies were undertaken to quantify the effects of transplantation of cotton (Gossypium hirsutum L.) seedlings during the early part of the growing season (March) and crop season (May) for potential realization of cotton productivity under the harsh weather of the southern part of Punjab province, Pakistan. Treatments, consisting of (a) two planting dates (March and May), and (b) two sowing methods (transplanting of seedlings and direct seeding), were arranged in a randomized complete-block design with four replications. Results showed that transplanting seedlings improved the productivity of cotton by 14.2% over direct seeding. Productivity was also increased substantially (34.8%) by planting during March over May sowing. The practice of planting cotton by transplanting seedlings and early sowing could be successfully adapted in areas where high temperatures coincide with the May planting and peak blooming periods in different cotton growing areas.

Cotton productivity enhanced through transplanting and early sowing / Aumento da produtividade do algodão por meio de transplante de mudas e semeadura precoce

Due to high temperatures in arid regions, cotton crop emergence and early establishment ofseedlings are adversely affected. Field studies were undertaken to quantify the effects of transplantation ofcotton (Gossypium hirsutum L.) seedlings during the early part of the growing season (March) and cropseason (May) for potential realization of cotton productivity under the harsh weather of the southern partof Punjab province, Pakistan. Treatments, consisting of (a) two planting dates (March and May), and (b)two sowing methods (transplanting of seedlings and direct seeding), were arranged in a randomizedcomplete-block design with four replications. Results showed that transplanting seedlings improved theproductivity of cotton by 14.2% over direct seeding. Productivity was also increased substantially (34.8%)by planting during March over May sowing. The practice of planting cotton by transplanting seedlings andearly sowing could be successfully adapted in areas where high temperatures coincide with the Mayplanting and peak blooming periods in different cotton growing areas.

Altas temperaturas, em torno de 48ºC, prevalecem durante os meses de maio na parte do sul da província de Punjab, no Paquistão. A esta temperatura a emergência da colheita de algodão e o estabelecimento precoce da plântula são adversamente afetados. Estudos de campo foram realizados paraquantificar os efeitos do transplante de mudas de algodão (Gossypium hirsutum L.) durante a primeira parteda estação de crescimento (março) e da safra (maio) para a realização potencial da produtividade do algodãosob condições adversas da parte sul do Punjab Província-Paquistão. Os tratamentos, consistindo em(a) duas datas de plantio (março e maio), e (b) dois métodos de semeadura (transplante de mudas e semeadura direta), foram dispostos em delineamento em blocos ao acaso com quatro repetições. Os resultados mostraram que o transplante de mudas melhorou a produtividade do algodão em 14,2% em relação à semeadura direta. A produtividade também aumentou substancialmente (34,8%) com o plantioem março sobre a semeadura de maio. A prática de plantio de algodão por transplante de mudas e sua semeadura precoce poderia ser adaptada com sucesso, onde a alta temperatura coincide com o tempo de plantio durante o mês de maio e períodos de florescimento máximo em diferentes áreas de cultivo de algodão.

Cotton productivity enhanced through transplanting and early sowing

Due to high temperatures in arid regions, cotton crop emergence and early establishment of seedlings are adversely affected. Field studies were undertaken to quantify the effects of transplantation of cotton (Gossypium hirsutum L.) seedlings during the early part of the growing season (March) and crop season (May) for potential realization of cotton productivity under the harsh weather of the southern part of Punjab province, Pakistan. Treatments, consisting of (a) two planting dates (March and May), and (b) two sowing methods (transplanting of seedlings and direct seeding), were arranged in a randomized complete-block design with four replications. Results showed that transplanting seedlings improved the productivity of cotton by 14.2% over direct seeding. Productivity was also increased substantially (34.8%) by planting during March over May sowing. The practice of planting cotton by transplanting seedlings and early sowing could be successfully adapted in areas where high temperatures coincide with the May planting and peak blooming periods in different cotton growing areas.

Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins.

Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomally synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study, a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target for the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible functional modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.

Single-step purification of cyclotides using affinity chromatography.

Cyclotides are considered promising scaffolds for drug development owing to their inherent host defence activities and highly stable structure, defined by the cyclic cystine knot. These proteins are expressed as complex mixtures in plants. Although several methods have been developed for their isolation and analysis, purification of cyclotides is still a lengthy process. Here, we describe the use of affinity chromatography for the purification of cyclotides using polyclonal IgG antibodies raised in rabbits against cycloviolacin O2 and immobilized on NHS-activated Sepharose columns. Cycloviolacin O2 was used as a model substance to evaluate the chromatographic principle, first as a pure compound and then in combination with other cyclotides, that is, bracelet cyclotide cycloviolacin O19 and Möbius cyclotide kalata B1, and in a plant extract. We demonstrate that single-step purification of cyclotides by affinity chromatography is possible but cross reactivity may occur between homologue cyclotides of the bracelet subfamily.

Outcomes Of Distal Femur Fracture Treated With Dynamic Condylar Screw.

BACKROUND: Implants for open reduction and internal fixation of distal femoral fracture includes angle blade plate, rush nails, enders nail and interlocking nails. But all these devices are technically demanding and less effective in providing inter-fragmentary compression in osteoporotic bones. These problems can be solved with dynamic condylar screw (DCS).The objective of the study was to determine the frequency of different outcomes of distal femoral fracture treated with dynamic condylar screw. METHODS: This case series study was carried out in the Department of Trauma & Orthopaedics, Ayub Teaching Hospital Abbottabad from 1st October 2014 to August 2015, after approval of the ethical committee of the institution. Data of all patients with distal femoral fractures aged 20-70 years, recruited through emergency, OPD or consultant clinic collected on a pro forma. Standard treatment of trauma was given to the patients. Detailed history was taken including the past medical and surgical history. Detailed examination including air-way, breathing and circulation, general physical examination and abdomino-pelvic examination was done in each patient. Investigations including urinalysis, haemoglobin %, full blood count, X-ray (both AP and lateral view) of the involved femur (including hip and knee) was done. RESULTS: Mean age of the patients was 43.18±14.647 ranging from 20 to 70 years. Mean duration of hospital stay in days was 2.21±1.111 ranging from 1 to 6 days. Patients' follow-up assessment after 4 months of surgery for union of femoral fracture treated with dynamic condylar screw was found in 96 (94.1%), wound infection was found in 7 (6.9%), knee stiffness was found in 21 (20.6%) and limb shortening was found in 7 (6.9%). CONCLUSIONS: Dynamic condylar screw is an easy, scientifically less difficult and satisfying method of treatment for fra.ctures of femur.

SUCCESSFUL TREATMENT OF MULTIDRUG-RESISTANT ACINETOBACTOR BAUMANII BLISTERS ON THE KNEE.

Acinetobacter baumannii (A. baumannii), nosocomial infections, especially those due to multi-drug resistant (MDR) strains, are increasingly detected. This study reports the case of a 50-year-old man with blisters on the right knee for 8 months, first admitted through the outpatient department for incisional biopsy. Microbiological and histo-pathological examination confirmed the diagnosis of blisters extending deeply up to the knee joint caused by MDR- A. baumannii. A broad spectrum antibiotic therapy was administered and later readjusted according to the results of microbiological culture and biopsy report. Intensive hemodynamic support was required. An extensive surgical debridement was promptly performed and repeated until complete control of the infection with intravenous colistins. Blisters were excised; wounds were dressed daily with chlorhexidine dressings and polymyxine-impregnated dressing. Wounds were finally covered with split-thickness skin grafts. The infection was overcome 120 days after admission. The graft take was 40%. Postoperative rehabilitation was required because of the functional limitation of lower limb movements at the knee joint. Follow-up at 8 months showed no functional deficit and an acceptable aesthetic result. AB-MDR affecting soft tissues is a life-threatening disease, especially in patients with poor immunity and limited access to health facilities, whose clinical diagnosis may sometimes be challenging. Early recognition and treatment represent the most important factors influencing survival.