Pediatric Acute Lymphoblastic Leukemia: Clinical Characteristics, Treatment Outcomes, and Prognostic Factors: 10 Years' Experience From a Low- and Middle-Income Country.

PURPOSE: Acute lymphoblastic leukemia (ALL) represents around 70% of pediatric leukemia. In high-income countries, the 5-year survival is above 90%, but survival in low- and middle-income countries is inferior. This study documents the treatment outcome and prognostic factors of pediatric ALL in Pakistan. MATERIALS AND METHODS: In this prospective cohort study, all newly diagnosed patients with ALL/lymphoblastic lymphoma from age 1 to 16 years enrolled between January 1, 2012, and December 31, 2021, were included. The treatment was based on the standard arm of the UKALL2011 protocol. RESULTS: Data from 945 patients with ALL, including 597 males (63.2%), were analyzed. The mean age at diagnosis was 5.73 ± 3.51 years. Pallor was the commonest presentation in 95.2% followed by fever in 84.2% of patients. The mean WBC count was 56.6 ± 103.4 × 109/L. Neutropenic fever followed by myopathy was the most common complication during induction. In univariate analysis, the high WBC count (P ≤ 0.001), intensive chemotherapy (P ≤ 0.001), malnutrition (P = .007), poor response to induction chemotherapy (P = .001), delayed presentation (P = .004), and use of steroids before chemotherapy (P = .023) significantly adversely affected overall survival (OS). The delayed presentation was the most significant prognostic factor in the multivariate analysis (P ≤ .002). After a median follow-up of 54.64 ± 33.80 months, the 5-year OS and disease-free survival (DFS) were 69.9% and 67.8%, respectively. CONCLUSION: In this largest cohort of childhood ALL from Pakistan, a high WBC count, malnutrition, delayed presentation, previous steroids use, intensive chemotherapy, and poor response to the induction chemotherapy were associated with decreased OS and DFS rates.

Structural and functional analyses of the echinomycin resistance conferring protein Ecm16 from Streptomyces lasalocidi.

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.

Comparative genomics of spike, envelope, and nucleocapsid protein of severe acute respiratory syndrome coronavirus 2.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) upsurge sprang up in Wuhan, China, in late December 2019. Objectives: Due to the exceptionally high mutation frequency, comparative genomics of viruses isolated throughout time and in various geographical locations are crucial. To better understand how SARS-CoV-2 heterogeneity has changed around the globe, this research was conducted. Methods: Nucleotide and protein sequences of SARS-CoV-2, SARS-CoV, and bat SARS-like CoV were extracted from the NCBI Virus database. The Wuhan SARS-CoV-2 variant was used as a reference. Molecular Evolutionary Genetics Study performed the phylogenetic analysis, while the Genome Detective Coronavirus Typing Tool performed the mutational analysis. Results: The evolutionary research has revealed that bats are the primary host for coronavirus evolution and the origin of the formation of SARS-CoV and SARS-CoV-2. Numerous mutations have been discovered in the spike, envelope, and nucleocapsid protein. Conclusions: The current research findings may have an implication that facilitates the development of prospective immunization candidates/small pharmacological compounds targeting COVID-19.

Biodiesel from Dodonaea Plant Oil: Synthesis and Characterization-A Promising Nonedible Oil Source for Bioenergy Industry.

In this work, Dodonaea oil was studied as a potential biodiesel source. Dodonaea (Dodonaea viscosa Jacq.) is an evergreen shrubby plant that thrives in tropical and subtropical conditions. The plant produces high-grade biodiesel in terms of both quantity and quality despite its naturally high fat content. In the transesterification followed by esterification reaction, varied ratios of oil to methanol, constant temperature (60°), reaction duration (1 h), and different catalyst concentrations (0.25-0.75% (w/w) were utilized. A maximum biodiesel yield of 90% was achieved. For fuel characteristic analysis, the prepared biodiesel was specified and compared to ASTM criteria. The chemical composition was verified using analytical techniques such as FT-IR and NMR spectroscopy. As a result of the foregoing, Dodonaea is considered a possible bioenergy source, particularly in the transport sector.

Comparative in vitro activity of selected antibacterial agents against Escherichia coli isolated from hospitalized patients suffering UTI.

Escherichia coli is the most studied among those bacteria causing urinary tract infections. This study was aimed to find out antibacterial activity and minimum inhibitory concentration of selected antibacterial agents against E. coli isolates of hospitalized UTI patients. The specimens were inoculated on Eosin Methylene Blue medium. E. coli isolates were identified via colonial morphology, biochemical testing and API-20 kit. The susceptibility pattern of antibacterial agents was determined applying disc diffusion method (Kirby-Bauer) and dilution tube method. Among all, 38.82% (n=158/407) specimens were positive for E. coli, while the rest showed either no growth or exhibited colonies other than E. coli. while observing the susceptibility pattern, Imipenem was found the most effective (73.42%) antibacterial agent, followed by nitrofurantoin (52.53%), cefpirome (44.94%) and tazobactam/ piperacillin (44.94%), whereas the E. coli isolates were highly resistant to sulfamethoxazole/trimethoprim (71.52%), followed by Amoxicillin-clavulanic acid (67.72%), nalidixic acid (66.46%) and Tobramycin (62.03%), when tested by disc diffusion method. The isolates were susceptible to cefpirome (39.87%) and tobramycin (39.87%) and resistant to sulfamethoxazole/trimethoprim (75.32%), followed by levofloxacin (61.39%), when tested by tube dilution method. The study concluded high degree of resistance against Sulfamethoxazole/trimethoprim, in contrast, cephalosporin and Imipenem exhibited good potency which can be recommended for UTI.

C-Reactive Protein and High-Sensitive Cardiac Troponins Correlate with Oxidative Stress in Valvular Heart Disease Patients.

Background: Valvular heart disease (VHD) is a major contributor to loss of physical function and longevity. Oxidative stress is one of the key causative factors involved in heart disease including VHD. Here, we aimed to illuminate the role and relation of oxidative stress to the VHD risk markers in the human population. Materials and Methods: 150 VHD patients and 103 healthy individuals as control were selected for the study and were divided into three groups: the aortic valve, mitral valve, and combined disease based on valvular calcification. Results: Our results demonstrated enhanced oxidative stress in the VHD condition, as we found elevated levels of reactive oxygen species (ROS) at the serum, supported by an increased level of thiobarbituric acid reactive substances (TBARs) in the cardiac valvular tissues of the VHD patients. In contrast, we experienced declined antioxidants including Super Oxide Dismutase (SOD), catalase (CAT), and peroxidase (POD) activities. Concurrently, increasing levels of C-reactive protein (CRP), high-sensitivity cardiac troponin I (hs-cTnI), and high-sensitivity cardiac troponin T (hs-cTnT) were detected in the aortic, mitral, and combined disease condition, suggesting a key association of oxidative stress to VHD conditions. Furthermore, regression analysis validated a key association between the impairment of the redox system (ROS and antioxidant enzyme activities) and VHD condition. Conclusion: Taken together, dysregulated oxidative stress contributes to the progression of VHD via positively correlating with CRP, hs-TnI, and hs-TnT level.

Three-Dimensional Structure Characterization and Inhibition Study of Exfoliative Toxin D From Staphylococcus aureus.

The Staphylococcus aureus exfoliative toxins (ETs) are the main toxins that produce staphylococcal scalded skin syndrome (SSSS), an abscess skin disorder. The victims of the disease are usually newborns and kids, as well as grown-up people. Five ETs namely, exfoliative toxins A, B, C, D, and E have been identified in S. aureus. The three-dimensional (3D) structure of exfoliative toxins A, B, C and E is known, while that of exfoliative toxin D (ETD) is still unknown. In this work, we have predicted the 3D structure of ETD using protein modeling techniques (software used for 3D structure modeling comprising the MODELLER 9v19 program, SWISS-Model, and I-TESSER). The validation of the build model was done using PROCHECK (Ramachandran plot), ERRAT2, and Verify 3D programs. The results from 3D modeling show that the build model was of good quality as indicated by a GMQE score of 0.88 and by 91.1% amino acid residues in the most favored region of the Ramachandran plot, the ERRAT2 quality factor of 90.1%, and a verify3D score of >0.2 for 99.59% of amino acid residues. The 3D structure analysis indicates that the overall structure of ETD is similar to the chymotrypsin-like serine protease fold. The structure is composed of 13 ß-strands and seven α-helices that fold into two well-defined six-strand ß-barrels whose axes are roughly perpendicular to each other. The active site residues include histidine-97, aspartic acid-147, and serine-221. This represents the first structure report of ETD. Structural comparison with the other ETs shows some differences, particularly in the loop region, which also change the overall surface charge of these toxins. This may convey variable substrate specificity to these toxins. The inhibition of these toxins by natural (2S albumin and flocculating proteins from Moringa oleifera seeds) and synthetic inhibitors (suramin) was also carried out in this study. The results from docking indicate that the inhibitors bind near the C-terminal domain which may restrict the movement of this domain and may halt the access of the substrate to the active site of this enzyme. Molecular dynamic simulation was performed to see the effect of inhibitor binding to the enzyme. This work will further elucidate the structure-function relationship of this enzyme. The inhibition of this enzyme will lead to a new treatment for SSSS.

Role of Lactobacillus plantarum 299v versus Placebo in symptomatic improvement of irritable bowel syndrome patients.

Objectives: To find the efficacy of lactobacillus plantarum 299v compared to placebo in symptomatic improvement of irritable bowel syndrome patients. METHODS: The randomised control trial was conducted at the Lady Reading Hospital, Peshawar, Pakistan, from July 20, 2014, to January 20, 2015, and comprised irritable bowel syndrome patients who were randomised into intervention group A treated with lactobacillus plantarum 299v and control group B treated with placebo. Symptoms, like abdominal pain, bloating and complete rectal emptying, were noted and compared between the groups. Data was analysed using SPSS 19. RESULTS: Of the 190 patients assessed, 120(63%) were included; 60(50%) in each of the two groups. After loss to follow-up, the study was completed by 55(91.7%) in group A and 53(88.3%) in group B. The mean age in group A was 37.53±9.02 and it was 34.40±11.23 in group B (p=0.652). The male-to-female ratio and irritable bowel syndrome type in both the groups was not significantly different (p>0.055). There was no significant difference in terms of relieving abdominal pain, bloating and rectal emptying between the groups (p>0.05). CONCLUSIONS: No significant efficacy was established in favour of treating in irritable bowel syndrome with lactobacillus plantarum 299v. Trials Registration: IRCT20200504047303N1.

Homozygous missense variant in POPDC3 causes recessive limb-girdle muscular dystrophy type 26.

BACKGROUND: Limb-girdle muscular dystrophy (LGMD) comprises a heterogeneous group of diseases, affecting different muscles, predominantly skeletal muscles and cardiac muscles of the body. LGMD is classified into two main subtypes A and B, which are further subclassified into eight dominant and thirty recessive subtypes. Three genes, namely POPDC1, POPDC2 and POPDC3, encode popeye domain-containing protein (POPDC), and the variants of POPDC1 and POPDC3 genes have been associated with LGMD. METHODS: In the present study, we performed whole-exome sequencing (WES) analysis on a single-family to investigate the hallmark features of LGMD. The results of WES were further confirmed by Sanger sequencing and 3D protein modeling was also conducted. RESULTS: WES data analysis and Sanger sequencing revealed a homozygous missense variant (c.460A>G; p.Lys154Glu) at a highly conserved amino acid position in the POPDC3. Mutations in the POPDC3 gene have been previously associated with recessive limb-girdle muscular dystrophy type 26. 3D protein modeling further suggested that the identified variant might affect the POPDC3 structure and proper function. CONCLUSIONS: The present study confirms the role of POPDC3 in LGMD, and will facilitate genetic counseling of the family to mitigate the risks of the carrier or affects on future pregnancies.

Inhibition of SARS-CoV-2 3CL Mpro by Natural and Synthetic Inhibitors: Potential Implication for Vaccine Production Against COVID-19.

COVID-19 has created a pandemic situation all over the world. It has spread in nearly every continent. Researchers all over the world are trying to produce an effective vaccine against this virus, however; no specific treatment for COVID-19 has been discovered -so far. The current work describes the inhibition study of the SARS-CoV-2 main proteinase or 3CL Mpro by natural and synthetic inhibitors, which include 2S albumin and flocculating protein from Moringa oleifera (M. oleifera) and Suramin. Molecular Docking study was carried out using the programs like AutoDock 4.0, HADDOCK2.4, patchdock, pardock, and firedock. The global binding energy of Suramin, 2S albumin, and flocculating proteins were -41.96, -9.12, and -14.78 kJ/mol, respectively. The docking analysis indicates that all three inhibitors bind at the junction of domains II and III. The catalytic function of 3CL Mpro is dependent on its dimeric form, and the flexibility of domain III is considered important for this dimerization. Our study showed that all three inhibitors reduce this flexibility and restrict their motion. The decrease in flexibility of domain III was further confirmed by analysis coming from Molecular dynamic simulation. The analysis results indicate that the temperature B-factor of the enzyme decreases tremendously when the inhibitors bind to it. This study will further explore the possibility of producing an effective treatment against COVID-19.

Putative second hit rare genetic variants in families with seemingly GBA-associated Parkinson's disease.

Rare variants in the beta-glucocerebrosidase gene (GBA1) are common genetic risk factors for alpha synucleinopathy, which often manifests clinically as GBA-associated Parkinson's disease (GBA-PD). Clinically, GBA-PD closely mimics idiopathic PD, but it may present at a younger age and often aggregates in families. Most carriers of GBA variants are, however, asymptomatic. Moreover, symptomatic PD patients without GBA variant have been reported in families with seemingly GBA-PD. These observations obscure the link between GBA variants and PD pathogenesis and point towards a role for unidentified additional genetic and/or environmental risk factors or second hits in GBA-PD. In this study, we explored whether rare genetic variants may be additional risk factors for PD in two families segregating the PD-associated GBA1 variants c.115+1G>A (ClinVar ID: 93445) and p.L444P (ClinVar ID: 4288). Our analysis identified rare genetic variants of the HSP70 co-chaperone DnaJ homolog subfamily B member 6 (DNAJB6) and lysosomal protein prosaposin (PSAP) as additional factors possibly influencing PD risk in the two families. In comparison to the wild-type proteins, variant DNAJB6 and PSAP proteins show altered functions in the context of cellular alpha-synuclein homeostasis when expressed in reporter cells. Furthermore, the segregation pattern of the rare variants in the genes encoding DNAJB6 and PSAP indicated a possible association with PD in the respective families. The occurrence of second hits or additional PD cosegregating rare variants has important implications for genetic counseling in PD families with GBA1 variant carriers and for the selection of PD patients for GBA targeted treatments.

The Anticancer Potential of Apigenin Via Immunoregulation.

Apigenin is an edible flavonoid widely distributed in natural plants, including most vegetables and fruits. Previous studies have revealed that apigenin possesses multiple biological functions by demonstrating antiinflammatory, anti-oxidative, anti-bacterial, anti-viral, anti-tumor and cardiovascular protective effects. Furthermore, recent progressions have disclosed a novel perspective of the anti-cancer roles of apigenin through its immunoregulatory functions. With the rapid progression of the groundbreaking strategies being developed for cancer immunotherapy, its immunoregulatory roles are being recognized as intriguing features of the multifaceted apigenin. However, the current understanding of this emerging role of apigenin still remains limited. Therefore, in the present review, recent advances on the immunoregulatory properties of apigenin in various diseases with a special focus on neoplasm, are summarized. Clinical strategies of cancer immunotherapy are briefly introduced and findings on apigenin linked to immunoregulatory roles in immunotherapy-associated aspects are brought together. The bioactivity, bioavailability, toxicity and potential of apigenin, to be considered as a therapeutic agent in anti-tumor immunotherapy, is discussed. Disclosed molecular mechanisms underlying the immunoregulatory roles of apigenin in cancer immunotherapy are also summarized. Based on findings from the literature, apigenin has the potential to serve as a prospective adjuvant for anti-cancer immunotherapy and warrants further investigations.

Synthesis and Structural Characterization of Biofuel From Cocklebur sp., Using Zinc Oxide Nano-Particle: A Novel Energy Crop for Bioenergy Industry.

This study is reporting the biofuel synthesis and characterization from the novel non-edible feedstock cocklebur seeds oil. The Cocklebur crop seeds oil was studied as a potential source for biofuel production based on the chemical, structural and fuel properties analysis. The oil expression and FFAs content in cocklebur crop was reported 37.2% and 0.47 gram KOH/g, using soxhlet apparatus and acid base titration method, respectively. The maximum conversion and yield of the cocklebur crop seeds non-edible oil to biofuel was pursued 93.33%, using transesterification process. The optimum protocol for maximum conversion yield was adjusted: 1:7 oil-methanol molar ratios, ZnO nano-particle concentration 0.2 gm (w/w), reaction temperature 60°C, and reaction time 45 min, respectively. ZnO nano-particle was prepared by a modified sol-gel method, using gelatin and the particle was XRD, TEM, XPS, and UV-vis spectroscopies. Qualitatively, the cocklebur crop synthesized biofuel was quantified and structurally characterized by GC/MS, FT-IR, NMR, and AAS spectroscopies. Quantitatively, the fuel properties of cocklebur crop biofuel was analyzed and compared with the international ASTM and EN standards.

Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles.

Latest advancement of omics technologies allows in-depth characterization of venom compositions. In the present work we present a proteomic study of two snake venoms of the genus Naja i.e., Naja naja (black cobra) and Naja oxiana (brown cobra) of Pakistani origin. The present study has shown that these snake venoms consist of a highly diversified proteome. Furthermore, the data also revealed variation among closely related species. High throughput mass spectrometric analysis of the venom proteome allowed to identify for the N. naja venom 34 protein families and for the N. oxiana 24 protein families. The comparative evaluation of the two venoms showed that N. naja consists of a more complex venom proteome than N. oxiana venom. Analysis also showed N-terminal acetylation (N-ace) of a few proteins in both venoms. To the best of our knowledge, this is the first study revealing this posttranslational modification in snake venom. N-ace can shed light on the mechanism of regulation of venom proteins inside the venom gland. Furthermore, our data showed the presence of other body proteins, e.g., ankyrin repeats, leucine repeats, zinc finger, cobra serum albumin, transferrin, insulin, deoxyribonuclease-2-alpha, and other regulatory proteins in these venoms. Interestingly, our data identified Ras-GTpase type of proteins, which indicate the presence of extracellular vesicles in the venom. The data can support the production of distinct and specific anti-venoms and also allow a better understanding of the envenomation and mechanism of distribution of toxins. Data are available via ProteomeXchange with identifier PXD018726.

The Sequence and Three-Dimensional Structure Characterization of Snake Venom Phospholipases B.

Snake venom phospholipases B (SVPLBs) are the least studied enzymes. They constitute about 1% of Bothrops crude venoms, however, in other snake venoms, it is present in less than 1%. These enzymes are considered the most potent hemolytic agent in the venom. Currently, no structural information is available about these enzymes from snake venom. To better understand its three-dimensional structure and mechanisms of envenomation, the current work describes the first model-based structure report of this enzyme from Bothrops moojeni venom named as B. moojeni phospholipase B (PLB_Bm). The structure model of PLB_Bm was generated using model building software like I-TESSER, MODELLER 9v19, and Swiss-Model. The build PLB_Bm model was validated using validation tools (PROCHECK, ERRAT, and Verif3D). The analysis of the PLB_Bm modeled structure indicates that it contains 491 amino acid residues that form a well-defined four-layer αßßα sandwich core and has a typical fold of the N-terminal nucleophile aminohydrolase (Ntn-hydrolase). The overall structure of PLB_Bm contains 18 ß-strands and 17 α-helices with many connecting loops. The structure divides into two chains (A and B) after maturation. The A chain is smaller and contains 207 amino acid residues, whereas the B chain is larger and contains 266 amino acid residues. The sequence and structural comparison among homologous snake venom, bacterial, and mammals PLBs indicate that differences in the length and sequence composition may confer variable substrate specificity to these enzymes. Moreover, the surface charge distribution, average volume, and depth of the active site cavity also vary in these enzymes. The present work will provide more information about the structure-function relationship and mechanism of action of these enzymes in snakebite envenomation.

REPORT - CYP2D6*4 null allele frequency in sixteen Pakistani ethnic groups.

CYP2D6 belongs to a family of Cytochrome P450 and is involved in metabolism of a number of commonly prescribed drugs. This study was designed to identify *4 allelic frequency of CYP2D6 in Pakistani population. The ethno-geographic variations in the CYP2D6 alleles are responsible for varied expression of this enzyme and thus influence the metabolic rate and efficacy of prescribed drugs. In total, 976 volunteers belonging to 16 different ethnic groups of Pakistan were screened for CYP2D6*4 polymorphism. The *4 allele was detected in all the ethnic groups with varied frequency ranging from 3.73%-13.64% and an overall average of 7.22% in different ethnic groups of the population. Maximum frequency was detected in northern Pakistani population including Meo (13.64%), Punjabi (11.96%) and Pathan (10.42%). Low frequency (<4%) of*4 polymorphism was observed in Kalash and Makrani groups, whereas an intermediate frequency (5-9%) was observed in all the other ethnic groups. The data indicates that despite ethnic diversity poor metabolizers in Pakistani population are expected to carry CYP2D6*4 allele at a relatively higher frequency than most other Asian populations. (Word count = 186).

Structure-Function Studies and Mechanism of Action of Snake Venom L-Amino Acid Oxidases.

Snake venom L-amino acid oxidases (SV-LAAOs) are the least studied venom enzymes. These enzymes catalyze the stereospecific oxidation of an L-amino acid to their corresponding α-keto acid with the liberation of hydrogen peroxide (H2O2) and ammonia (NH3). They display various pathological and physiological activities including induction of apoptosis, edema, platelet aggregation/inhibition, hemorrhagic, and anticoagulant activities. They also show antibacterial, antiviral and leishmanicidal activity and have been used as therapeutic agents in some disease conditions like cancer and anti-HIV drugs. Although the crystal structures of six SV-LAAOs are present in the Protein Data Bank (PDB), there is no single article that describes all of them in particular. To better understand their structural properties and correlate it with their function, the current work describes structure characterization, structure-based mechanism of catalysis, inhibition and substrate specificity of SV-LAAOs. Sequence analysis indicates a high sequence identity (>84%) among SV-LAAOs, comparatively lower sequence identity with Pig kidney D-amino acid oxidase (<50%) and very low sequence identity (<24%) with bacterial LAAOs, Fugal (L-lysine oxidase), and Zea mays Polyamine oxidase (PAAO). The three-dimensional structure of these enzymes are composed of three-domains, a FAD-binding domain, a substrate-binding domain and a helical domain. The sequence and structural analysis indicate that the amino acid residues in the loops vary in length and composition due to which the surface charge distribution also varies that may impart variable substrate specificity to these enzymes. The active site cavity volume and its average depth also vary in these enzymes. The inhibition of these enzymes by synthetic inhibitors will lead to the production of more potent antivenoms against snakebite envenomation.

Determining the Structures of the Snake and Spider Toxins by X-Rays.

Snake and spider venom is a complex mixture that contains proteins, peptides, and small organic and inorganic compounds. In contrast to spider venom, snake venom proteins are well known both functionally and structurally. This work describes methods for purification and crystallization of snake and spider venom toxins and their three-dimensional structure determination by X-ray crystallography.

The Sequence and a Three-Dimensional Structural Analysis Reveal Substrate Specificity Among Snake Venom Phosphodiesterases.

(1) Background. Snake venom phosphodiesterases (SVPDEs) are among the least studied venom enzymes. In envenomation, they display various pathological effects, including induction of hypotension, inhibition of platelet aggregation, edema, and paralysis. Until now, there have been no 3D structural studies of these enzymes, thereby preventing structure-function analysis. To enable such investigations, the present work describes the model-based structural and functional characterization of a phosphodiesterase from Crotalusadamanteus venom, named PDE_Ca. (2) Methods. The PDE_Ca structure model was produced and validated using various software (model building: I-TESSER, MODELLER 9v19, Swiss-Model, and validation tools: PROCHECK, ERRAT, Molecular Dynamic Simulation, and Verif3D). (3) Results. The proposed model of the enzyme indicates that the 3D structure of PDE_Ca comprises four domains, a somatomedin B domain, a somatomedin B-like domain, an ectonucleotide pyrophosphatase domain, and a DNA/RNA non-specific domain. Sequence and structural analyses suggest that differences in length and composition among homologous snake venom sequences may account for their differences in substrate specificity. Other properties that may influence substrate specificity are the average volume and depth of the active site cavity. (4) Conclusion. Sequence comparisons indicate that SVPDEs exhibit high sequence identity but comparatively low identity with mammalian and bacterial PDEs.

Segregation and potential functional impact of a rare stop-gain PABPC4L variant in familial atypical Parkinsonism.

Atypical parkinsonian disorders (APDs) comprise a group of neurodegenerative diseases with heterogeneous clinical and pathological features. Most APDs are sporadic, but rare familial forms have also been reported. Epidemiological and post-mortem studies associated APDs with oxidative stress and cellular protein aggregates. Identifying molecular mechanisms that translate stress into toxic protein aggregation and neurodegeneration in APDs is an active area of research. Recently, ribonucleic acid (RNA) stress granule (SG) pathways were discussed to be pathogenically relevant in several neurodegenerative disorders including APDs. Using whole genome sequencing, mRNA expression analysis, transfection assays and cell imaging, we investigated the genetic and molecular basis of a familial neurodegenerative atypical parkinsonian disorder. We investigated a family with six living members in two generations exhibiting clinical symptoms consistent with atypical parkinsonism. Two affected family members suffered from parkinsonism that was associated with ataxia. Magnetic resonance imaging (MRI) of these patients showed brainstem and cerebellar atrophy. Whole genome sequencing identified a heterozygous stop-gain variant (c.C811T; p.R271X) in the Poly(A) binding protein, cytoplasmic 4-like (PABPC4L) gene, which co-segregated with the disease in the family. In situ hybridization showed that the murine pabpc4l is expressed in several brain regions and in particular in the cerebellum and brainstem. To determine the functional impact of the stop-gain variant in the PABPC4L gene, we investigated the subcellular localization of PABPC4L in heterologous cells. Wild-type PABPC4L protein localized predominantly to the cell nucleus, in contrast to the truncated protein encoded by the stop-gain variant p.R271X, which was found homogeneously throughout the cell. Interestingly, the wild-type, but not the truncated protein localized to RasGAP SH3 domain Binding Protein (G3BP)-labeled cytoplasmic granules in response to oxidative stress induction. This suggests that the PABPC4L variant alters intracellular distribution and possibly the stress granule associated function of the protein, which may underlie APD in this family. In conclusion, we present genetic and molecular evidence supporting the role of a stop-gain PABPC4L variant in a rare familial APD. Our data shows that the variant results in cellular mislocalization and inability of the protein to associate with stress granules.