Distinct melanization pathways in the mosquito Aedes aegypti.

Serine protease cascades are involved in blood coagulation and immunity. In arthropods, they regulate melanization, which plays an important role in immune defense and wound healing. However, the mechanisms underlying melanization pathways are not completely characterized. We found that in the mosquito Aedes aegypti, there are two distinct melanization activation pathways carried out by different modules of serine proteases and their specific inhibitors serpins. Immune melanization proteases (IMP-1 and IMP-2) and Serpin-1 mediate hemolymph prophenoloxidase cleavage and immune response against the malaria parasite. Tissue melanization, exemplified by the formation of melanotic tumors, is controlled by tissue melanization protease (CLIPB8), IMP-1, and Serpin-2. In addition, serine proteases CLIPB5 and CLIPB29 are involved in activation of Toll pathway by fungal infection or by infection-independent manner, respectively. Serpin-2 is implicated in the latter activation of Toll pathway. This study revealed the complexity underlying melanization and Toll pathway in mosquitoes.

Novel fructose bisphosphatase 1 gene mutation presenting as recurrent episodes of vomiting in an Indian child.

Fructose-1, 6-bisphosphatase 1 (FBP1) deficiency is an autosomal recessive disorder of gluconeogenesis resulting in severe and recurrent life-threatening episodes of hypoglycemia and lactic acidosis in infancy. We report a 16 month-old girl who presented with recurrent episodes of vomiting, rapid breathing, lactic acidosis, hyperuricemia, and hypertriglyceridemia. Genetic analysis revealed a novel compound heterozygous mutation in FBP1 gene confirming the diagnosis of FBP1 deficiency. The patient was managed with treatment of acute episodes and preventive long-term dietary modifications. Long-term prognosis of FBP1 deficiency is excellent underlining the importance of early recognition of clinical signs, prompt diagnosis, and avoidance of fasting in this disease. FBP1 gene mutations have been described from various ethnic backgrounds, but there is limited data available from Indian population, hence the importance of this case.

Site-Selective Oxidative Coupling Reactions for the Attachment of Enzymes to Glass Surfaces through DNA-Directed Immobilization.

Enzymes are able to maintain remarkably high selectivity toward their substrates while still retaining high catalytic rates. By immobilizing enzymes onto surfaces we can heterogenize these biological catalysts, making it practical to study, use, and combine them in an easily controlled system. In this work, we developed a platform that allows for the simple and oriented immobilization of proteins through DNA-directed immobilization. First, we modified a glass surface with single-stranded DNA. We then site-selectively attached the complementary DNA strand to the N-terminus of a protein. Both DNA modifications were carried out using an oxidative coupling strategy, and the DNA strands served as easily tunable and reversible chemical handles to hybridize the protein-DNA conjugates onto the surface. We have used the aldolase enzyme as a model protein to conduct our studies. We characterized each step of the protein immobilization process using fluorescent reporters as well as atomic force microscopy. We also conducted activity assays on the surfaces with DNA-linked aldolase to validate that, despite being modified with DNA and undergoing subsequent immobilization, the enzyme was still able to retain its catalytic activity and the surfaces were reusable in subsequent cycles.

How sesquiterpenes modulate signaling cascades in cancers.

Data obtained from high-throughput technologies has started to shed light on the interplay between signal transduction cascades and chromatin modifications thus adding another layer of complexity to the already complex regulation of the protein network. Based on the insights gleaned from almost a decade of research, it has now been convincingly revealed that sesquiterpenes effectively modulated different intracellular signaling cascades in different cancers. In this review we summarize how sesquiterpenes mediated Wnt, Shh, Notch and TRAIL induced signaling cascades.

Optimization and expansion of a site-selective N-methylpyridinium-4-carboxaldehyde-mediated transamination for bacterially expressed proteins.

Site-selective bioconjugation methods are valuable because of their ability to confer new properties to proteins by the chemical attachment of specific functional groups. Well-defined bioconjugates obtained through these methods have found utility for the study of protein function and the creation of protein-based materials. We have previously reported a protein modification strategy to modify the N-terminus of peptides and proteins using N-methylpyridinium-4-carboxaldehyde benzenesulfonate (Rapoport's salt, RS) as a transamination reagent, which oxidizes the N-terminal amino group to provide a uniquely reactive aldehyde or ketone. This functional handle can subsequently be modified with an alkoxyamine reagent of choice. Previous work had found glutamate terminal sequences to be highly reactive toward RS-mediated transamination. However, proteins of interest are often recombinantly expressed in E. coli, where the expression of a glutamate-terminal protein is rendered difficult because the N-terminal methionine derived from the start codon is not cleaved when Glu is in the second position. In this work, we describe a way to overcome this difficulty via the insertion of a Factor Xa proteolytic cleavage site to acquire the optimal glutamate residue at the N-terminus. Additionally, we present studies on alternative high-yielding sequences containing N-terminal residues that can be expressed directly. We have used site-directed mutagenesis to validate these findings on a model cellulase enzyme, an endoglucanase from the thermophilic Pyrococcus horikoshii. Activity assays performed with these mutants show that RS transamination and subsequent modification with alkoxyamines have no negative impact on cellulolytic ability.

A cohort study of MFN2 mutations and phenotypic spectrums in Charcot-Marie-Tooth disease 2A patients.

Charcot-Marie-Tooth disease 2A (CMT2A) is the most common axonal form of peripheral neuropathy caused by a defect in the mitofusin 2 (MFN2) gene, which encodes an outer mitochondrial membrane GTPase. MFN2 mutations result in a large range of phenotypes. This study analyzed the prevalence of MFN2 mutation in Korean families with their assorted phenotypes (607 CMT families and 160 CMT2 families). Direct sequencing of the MFN2 coding exons or whole-exome sequencing has been applied to identify causative mutations. A total of 21 mutations were found in 36 CMT2 families. Comparative genotype-phenotype correlations impacting severity, onset age, and specific symptoms were assessed. Most mutations were seen in the GTPase domain (∼86%). A deletion mutation found in the transmembrane helices is reported for the first time, as well as five novel mutations at other domains. MFN2 mutations made up 5.9% of total CMT families, whereas 22.9% in CMT2 families, of which 27.8% occurred de novo. Interestingly, patient phenotypes ranged from mild to severe even for the same mutation, suggesting other factors influenced phenotype and penetrance. This CMT2A cohort study will be useful for molecular diagnosis and treatment of axonal neuropathy.

Site-specific protein transamination using N-methylpyridinium-4-carboxaldehyde.

The controlled attachment of synthetic groups to proteins is important for a number of fields, including therapeutics, where antibody-drug conjugates are an emerging area of biologic medicines. We have previously reported a site-specific protein modification method using a transamination reaction that chemoselectively oxidizes the N-terminal amine of a polypeptide chain to a ketone or an aldehyde group. The newly introduced carbonyl can be used for conjugation to a synthetic group in one location through the formation of an oxime or a hydrazone linkage. To expand the scope of this reaction, we have used a combinatorial peptide library screening platform as a method to explore new transamination reagents while simultaneously identifying their optimal N-terminal sequences. N-Methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport's salt, RS) was identified as a highly effective transamination reagent when paired with glutamate-terminal peptides and proteins. This finding establishes RS as a transamination reagent that is particularly well suited for antibody modification. Using a known therapeutic antibody, herceptin, it was demonstrated that RS can be used to modify the heavy chains of the wild-type antibody or to modify both the heavy and the light chains after N-terminal sequence mutation to add additional glutamate residues.

Addition of transglutaminase enzyme in camel milk yoghurt to increase its sensorial aspects.

Camel milk was obtained from A-block UVAS Ravi Campus Pattoki. After pasteurization at 72 °C (15 sec) it was cooled to 42 °C, then glutathione treated transglutaminase enzyme was added with the concentration of 0.5 g/300 mL, 1 g/300 mL, 1.5 g/300 mL, 2 g/300 mL while control sample with the addition of 1.5 g/300 mL gelatin. Then inoculation of milk was done with standard cultures of Yoghurt Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus at the rate of 2% for 3-4 hours at 42 °C. Samples were stored at 4 °C and were analyzed on 1st day and 28th day of storage. In our findings, there was slight increase in sensorial properties of all the samples. It was also observed that syneresis was reduced with the increase of enzyme quantity.

Genetic variation between hybrid (Labeo rohita ♂, Cirrhinus mrigala ♀ and Labeo rohita) by RAPD marker.

The present work was to study the genetic variability between the major carps Labeo rohita and Cirrhinus mrigala and their hybrids of L. rohita (male♂) and C. mrigala (female♀). Genetic variability was studied by employing RAPD molecular markers. 25 samples of each target species having different sizes with the same age group for the determination of interspecific variation were collected. The morphometric parameters such as body weight, total length, tail length, and lengths of dorsal and anal fins of each individual were recorded and results showed that wet body weight, total length, dorsal fin, anal fin, and tail fin length are positively correlated and then the DNA was extracted using the inorganic salt-based method and conformed by Gel electrophoresis. Twenty-four arbitrary decamer primers were used to get species-specific RAPD analysis Distinct and highly reproducible RAPD profiles with significant genetic variability was detected among species. Only five primers showed amplification. The RAPAD primer OPB-05 produced a total of seven bands out of these 5 monomorphic and 2 polymorphic, so in this case, the percentage polymorphism was 28.57%. The Hybrid show more than a 50% difference from the Labeo rohita. This shows that the Hybrid more resembles C.mrigala. Phylogenetic analysis demonstrated that hybrid (L. rohita ♂ X Cirrhinus mrigala ♀) is the closest to C. mrigala and the farthest from L. rohita. Overall data are presented concerning the applications of RAPD markers for hybrid identification, genetic diversity assessment, and studying taxonomic relationships at a molecular level.

Double Trouble: An Unusual Keratitis Case of Herpes Simplex Virus and Varicella-Zoster Virus Co-infection.

PURPOSE: The aim of this study was to describe a case of herpes simplex virus (HSV) and varicella-zoster virus (VZV) corneal co-infection in a patient with systemic immunosuppression. METHODS: A 77-year-old White man who was recently administered pembrolizumab present with reduction in visual acuity in his left eye from 20/25 to 20/50. There was a known history of ocular HSV keratitis. Slit-lamp examination showed superficial dendritic lesions suggestive of VZV. RESULTS: Viral polymerase chain reaction testing was positive for both HSV and VZV, confirming clinical diagnosis of VZV keratitis in the setting of recurrent HSV keratitis. The infection responded to treatment with topical trifluridine. Two months later, he had another episode of keratitis based on his symptoms reported through telephone encounter which resolved with trifluridine. Unfortunately, the patient committed suicide 4 months after onset. CONCLUSIONS: This is the first case of keratitis with HSV and VZV co-infection likely related to systemic immunosuppression. Clinicians should have a high suspicion for viral co-infections in the setting of systemic immunosuppression.

Investigation of roost composition of passerine birds in different environmental conditions.

The majority of the birds in different habitats are stressed due to alteration in multiple climate factors contributing to their loss. The present study has been planned to find the roosts composition of passerine birds in different major and sub-habitats of Punjab, Pakistan. In Faisalabad, of the four species, the higher number of exits was almost comparable, while Passer domesticus and Pastor roseus were more abundant than Tachycinet bicolor and Lanius cristatus. For the three remaining birds, total exits and returns were 180 for P. roseus, 181 for T. bicolor, and 179 for L. cristatus, respectively. Considering the exits in morning hours, a total of 314, 256, 246 and 210, were recorded from Sheikhupura. In Khanewal, of the four species, the highest exits and returns were that of P. domesticus (407; 451), followed by that of the P. roseus (273; 336), T. bicolor (242; 319) and L. cristatus (220; 397). The temperature imposed serious effects on roost exits for the four birds. The varied P-values which were higher (< 0.001***, < 0.001***, 0.002 **, <0.001***) appeared to limit the roost exits for them. Nonetheless, the impact of relative humidity exerted a strong influence on the T. bicolor (0.003**). In roosts return, it was seen that roost returns were even likely in warm temperatures and precipitation did not impose seriously on returns, and even in light rainfall. Nonetheless, relative humidity (RH) strongly impacted the sparrow. The T. bicolor and L. cristatus were adversely affected with the slopes (1.37) and (2.06), indicated with each percentage increase of relative humidity, and slope variations became least.

Assessment of essential minerals and physico-chemical analysis of floral origins fresh honey produced by Apis mellifera.

Honey is one of the best nutritious substances in the world, having different services in the body functions regulation. Ten elements (K, Na, Ca, Co, Cr, Mn, Mo, Ni, Pb, Se) from honey samples were analyzed from 80 different locations of Punjab and ten floras. The aim of the present study was to determine the quality and quantity of minerals and Physico-chemical analysis in honey. A flame photometer was used to measure the concentration of major minerals (K, Ca and Na). The concentration of micro minerals (Co, Cr, Mn, Mo, Ni, Pb and Se) was analyzed using Atomic Absorption Spectrometer. The concentration of macro-elements obtained was as follow (in ppm): K (166-1732), Na (107-418) and Ca (07-99), while the concentration of microelements (in ppm) Co (1-2), Cr (>1), Mn (<1), Mo (1.818), Ni (1.911), Pb (<1) and Se (1.968). The most abundant minerals were potassium, calcium and sodium, ranging between 396-810.5, 17.5-640.63 and 169.88-238.62 ppm, respectively. However, the trace mineral elements of honey were obtained in the order of decreasing Se > Co > Ni > Pb > Cr > Mo > Mn. The findings showed that all the heavy metals like Co, Cr, Ni and Pb were present in trace amounts and close to International Honey Quality Standard. The result of given honey samples represented highest value of moisture (31.23%), color (80 mm pfund), pH (8.23), acidity (72.02 meq/kg), electrical conductivity (0.85 ms/cm) and ash contents (0.83%).

Mosquito RUNX4 in the immune regulation of PPO gene expression and its effect on avian malaria parasite infection.

Prophenoloxidases (PPOs) are key enzymes of the melanization reaction, which is a prominent defense mechanism of arthropods. The mosquito Aedes aegypti has ten PPO genes in the genome, four of which (PPO1, PPO3, PPO5, and PPO8) were expressed in response to microbial infection. Cactus depletion resulted in transcriptional activation of these four genes, suggesting this up-regulation to be under the control of the Toll pathway. The silencing of Cactus also led to developmental arrest and death of the avian malaria parasite, Plasmodium gallinaceum. We discovered that RUNT-related transcription factor 4 (RUNX4), the orthologue of Drosophila Lozenge, bound to the RUNT binding motif in the promoter of mosquito PPO genes and stimulated the expression of Drosophila PPO-A1 and PPO3 in S2 cell line. The immune effects caused by Cactus depletion were eliminated by double knockdown of Cactus/RUNX4. These findings suggest that RUNX4 regulates PPO gene expression under the control of the Toll pathway and plays a critical role in restricting parasite development.

Using Virtual Simulations in Online Laboratory Instruction and Active Learning Exercises as a Response to Instructional Challenges during COVID-19.

The onset of the COVID-19 pandemic in the spring of 2020 thrust instructors into a world of frenzy, presenting unique challenges to delivering course content. A particular challenge was determining suitable substitutes for wet lab experiments that are often comprised in science labs. Recognizing that this problem was not short-term, I started to look into virtual substitutions to be implemented in the 2020-2021 academic year. Virtual simulations can replace labs, be incorporated as pre-lab assignments, or used as active-learning or experiential learning exercises in a traditional classroom setting while providing low-cost, safe, and acceptable solutions to the current problem. Virtual simulations were examined on different platforms, including Labster, McGraw Hill Connect Virtual Labs, HHMI BioInteractive, Learn.Genetics, Virtual Interactive Bacteriology Laboratory, and Biology Corner. The goal was to provide faculty around the world with a reference list of virtual simulations that are aligned to specific AAAS and ASM student learning outcomes. These simulations are discussed in terms of content, features, and advantages of use. A list of lab exercises aligned to biology courses (microbiology, genetics, and cell biology) is also provided.

Development of chitosan based microencapsulated spray dried powder of tuna fish oil: oil load impact and oxidative stability.

The impact of fish oil concentration on the oxidative stability of microcapsules through the spray drying process using chitosan and maltodextrin as wall material was studied. Emulsions were prepared with different Tuna fish oil (TFO) content (TFO-10%, TFO20%, TF030% TF0-40%) while wall material concentration was kept constant. Microencapsulated powder resulting from emulsion prepared with high fish oil load have high moisture content, wettability, total oil and low encapsulation efficiency, hygroscopicity and bulk tapped density. Oxidative stability was evaluated periodically by placing microcapsules at room temperature. Microcapsules prepared with TFO-10% presented high oxidative stability in terms of peroxide value (2.94±0.04) and anisidine value (1.54±0.02) after 30 days of storage. It was concluded that optimal amounts of fish oil for microencapsulation are 10% and 20% using chitosan and maltodextrin that extended its shelf life during study period.

Novel finding of atrophic extraocular muscles in Loeys-Dietz syndrome: a case report and review of the literature.

A 19-year-old man with Loeys-Dietz syndrome and right exotropic Duane syndrome after bilateral lateral rectus recessions at age 22 months presented with recurrent progressive exotropia 17 years after his initial surgery. Surgical correction was aborted intraoperatively when extreme atrophy of the right medial rectus, lateral rectus, and superior rectus muscles was observed, later corroborated by orbital magnetic resonance imaging.

Teaching in the Time of COVID-19: Creation of a Digital Internship to Develop Scientific Thinking Skills and Create Science Literacy Exercises for Use in Remote Classrooms.

The extreme academic and social disruption caused by COVID-19 in the spring and summer of 2020 led to the loss of many student internships. We report here our creation of a novel internship for students majoring in the biological sciences. Student interns worked together to systematically categorize multiple episodes of This Week in Microbiology (TWiM). They annotated episodes by labeling relevant ASM fundamental curricular guidelines and the microbiology techniques described in several podcast episodes. Interns worked together, which advanced their written and oral communication skills while improving their scientific thinking skills. Faculty then enhanced each annotation by adding short figure-reading exercises that can be used in a variety of educational settings to teach science literacy. When surveyed, students reported greater confidence in analyzing and interpreting results from a variety of microbiological methods, improved communication of fundamental microbiology concepts in written and oral form, and enhanced ability to collaborate with others. Combined, this digital internship provided a unique opportunity for students to develop critical technical and scientific thinking skills and generated useful open education resources for teaching general microbiology in the form of annotated podcasts.

Amelioration potential of Moringa oleifera extracts against sodium arsenate induced embryotoxicity and genotoxicity in mouse (Mus musculus).

Previous studies have suggested that arsenic crosses the placenta and affects the fetus development. The study under consideration aims to show comparative ameliorative effect of Moringa oleifera leaf and flower extracts against sodium arsenate induced fetus toxicity of mice. Pregnant mice (N=44) were kept in lab and divided into eleven group from (A to K) and were orally administered the doses 6 mg/kg, 12 mg/kg for sodium arsenate, 150 mg/kg and 300 mg/kg for Moringa oleifera leaf extracts (MOLE) and 150 mg/kg and 300 mg/kg for Moringa oleifera flower extracts (MOFE) comparing with control. The investigation revealed evident reduction in the fetuses weight, hind limb, fore limb, tail and snout length, crown rump and head circumferences well as malformations in tail, feet, arms, legs, skin and eyes in the negative control group (only administered with sodium arsenate). Co-administration of sodium arsenate with MOLE and MOFE ameliorate the reversed effect of sodium arsenate on the shape, length, body weight and DNA damage of fetus significantly at 95% confidence interval. However, Moringa oleifera leaf extract showed more significant results in comparison to Moringa oleifera flower extract. Hence concluded that Moringa oleifera leaf extract ameliorated the embryo toxic effects of sodium arsenate and can be used against environmental teratogens.