Single-cell investigative genetics: Single-cell data produces genotype distributions concentrated at the true genotype across all mixture complexities.

In the absence of a suspect the forensic aim is investigative, and the focus is one of discerning what genotypes best explain the evidence. In traditional systems, the list of candidate genotypes may become vast if the sample contains DNA from many donors or the information from a minor contributor is swamped by that of major contributors, leading to lower evidential value for a true donor's contribution and, as a result, possibly overlooked or inefficient investigative leads. Recent developments in single-cell analysis offer a way forward, by producing data capable of discriminating genotypes. This is accomplished by first clustering single-cell data by similarity without reference to a known genotype. With good clustering it is reasonable to assume that the scEPGs in a cluster are of a single contributor. With that assumption we determine the probability of a cluster's content given each possible genotype at each locus, which is then used to determine the posterior probability mass distribution for all genotypes by application of Bayes' rule. A decision criterion is then applied such that the sum of the ranked probabilities of all genotypes falling in the set is at least 1-α. This is the credible genotype set and is used to inform database search criteria. Within this work we demonstrate the salience of single-cell analysis by performance testing a set of 630 previously constructed admixtures containing up to 5 donors of balanced and unbalanced contributions. We use scEPGs that were generated by isolating single cells, employing a direct-to-PCR extraction treatment, amplifying STRs that are compliant with existing national databases and applying post-PCR treatments that elicit a detection limit of one DNA copy. We determined that, for these test data, 99.3% of the true genotypes are included in the 99.8% credible set, regardless of the number of donors that comprised the mixture. We also determined that the most probable genotype was the true genotype for 97% of the loci when the number of cells in a cluster was at least two. Since efficient investigative leads will be borne by posterior mass distributions that are narrow and concentrated at the true genotype, we report that, for this test set, 47,900 (86%) loci returned only one credible genotype and of these 47,551 (99%) were the true genotype. When determining the LR for true contributors, 91% of the clusters rendered LR>1018, showing the potential of single-cell data to positively affect investigative reporting.

Designing Configurable Soft Microgelsomes as a Smart Biomimetic Protocell.

Self-assembly is an intriguing aspect of primitive cells. The construction of a semipermeable compartment with a robust framework of soft material capable of housing an array of functional components for chemical changes is essential for the fabrication of synthetic protocells. Microgels, loosely cross-linked polymer networks, are suitable building blocks for protocell capsule generation due to their porous structure, tunable properties, and assembly at the emulsion interface. Here, we present an interfacial assembly of microgel-based microcompartments (microgelsomes, MGC) that are defined by a semipermeable, temperature-responsive elastic membrane formed by densely packed microgels in a monolayer. The water-dispersible microgelsomes can thermally shuttle between 10 and 95 °C while retaining their structural integrity. Importantly, the microgelsomes exhibited distinct properties of protocells, such as cargo encapsulation, semipermeable membrane, DNA amplification, and membrane-gated compartmentalized enzymatic cascade reaction. This versatile approach for the construction of biomimetic microcompartments augments the protocell library and paves the way for programmable synthetic cells.

Advances in paper based isothermal nucleic acid amplification tests for water-related infectious diseases.

Water-associated or water-related infectious disease outbreaks are caused by pathogens such as bacteria, viruses, and protozoa, which can be transmitted through contaminated water sources, poor sanitation practices, or insect vectors. Low- and middle-income countries bear the major burden of these infections due to inadequate hygiene and subpar laboratory facilities, making it challenging to monitor and detect infections in a timely manner. However, even developed countries are not immune to these diseases, as inadequate wastewater management and contaminated drinking water supplies can also contribute to disease outbreaks. Nucleic acid amplification tests have proven to be effective for early disease intervention and surveillance of both new and existing diseases. In recent years, paper-based diagnostic devices have made significant progress and become an essential tool in detecting and managing water-associated infectious diseases. In this review, we have highlighted the importance of paper and its variants as a diagnostic tool and discussed the properties, designs, modifications, and various paper-based device formats developed and used for detecting water-associated pathogens.

Bactericidal and biocatalytic temperature responsive microgel based self-cleaning membranes for water purification.

The present study focuses on creating an antimicrobial and biocatalytic smart gating membrane by synthesizing unique core-shell microgels. The core-shell microgels are synthesized by grafting short chains of poly(ethylenimine) (PEI) onto a poly((N-isopropyl acrylamide)-co-glycidyl methacrylate)) (P(NIPAm-co-GMA)) core. Subsequently, the produced microgels are utilized as a substrate for synthesizing and stabilizing silver nanoparticles (Ag NPs) through an in-situ approach. These Ag NPs immobilized microgels are then suction filtered over a polyethylene terephthalate (PET) track-etched support to create cross-linked composite microgel membranes (CMMs). After structural and permeation characterization of the prepared CMMs, the laccase enzyme is then covalently grafted to the surface of the membrane and tested for its effectiveness in degrading Reactive red-120 dye. The laccase immobilized biocatalytic CMMs show effective degradation of the Reactive red-120 by 71%, 48%, and 34% at pH 3, 4, and 5, respectively. Furthermore, the immobilized laccase enzyme showed better activity and stability in terms of thermal, pH, and storage compared to the free laccase, leading to increased reusability. The unique combination of Ag NPs and laccase on a thermoresponsive microgel support resulted in a responsive self-cleaning membrane with excellent antimicrobial and dye degradation capabilities for environmentally friendly separation technology.

Biocatalytic self-assembled synthetic vesicles and coacervates: From single compartment to artificial cells.

Compartmentalization is an intrinsic feature of living cells that allows spatiotemporal control over the biochemical pathways expressed in them. Over the years, a library of compartmentalized systems has been generated, which includes nano to micrometer sized biomimetic vesicles derived from lipids, amphiphilic block copolymers, peptides, and nanoparticles. Biocatalytic vesicles have been developed using a simple bag containing enzyme design of liposomes to multienzymes immobilized multi-vesicular compartments for artificial cell generation. Additionally, enzymes were also entrapped in membrane-less coacervate droplets to mimic the cytoplasmic macromolecular crowding mechanisms. Here, we have discussed different types of single and multicompartment systems, emphasizing their recent developments as biocatalytic self-assembled structures using recent examples. Importantly, we have summarized the strategies in the development of the self-assembled structure to improvise their adaptivity and flexibility for enzyme immobilization. Finally, we have presented the use of biocatalytic assemblies in mimicking different aspects of living cells, which further carves the path for the engineering of a minimal cell.

Nature Inspired Multienzyme Immobilization: Strategies and Concepts.

In a biological system, the spatiotemporal arrangement of enzymes in a dense cellular milieu, subcellular compartments, membrane-associated enzyme complexes on cell surfaces, scaffold-organized proteins, protein clusters, and modular enzymes have presented many paradigms for possible multienzyme immobilization designs that were adapted artificially. In metabolic channeling, the catalytic sites of participating enzymes are close enough to channelize the transient compound, creating a high local concentration of the metabolite and minimizing the interference of a competing pathway for the same precursor. Over the years, these phenomena had motivated researchers to make their immobilization approach naturally realistic by generating multienzyme fusion, cluster formation via affinity domain-ligand binding, cross-linking, conjugation on/in the biomolecular scaffold of the protein and nucleic acids, and self-assembly of amphiphilic molecules. This review begins with the discussion of substrate channeling strategies and recent empirical efforts to build it synthetically. After that, an elaborate discussion covering prevalent concepts related to the enhancement of immobilized enzymes' catalytic performance is presented. Further, the central part of the review summarizes the progress in nature motivated multienzyme assembly over the past decade. In this section, special attention has been rendered by classifying the nature-inspired strategies into three main categories: (i) multienzyme/domain complex mimic (scaffold-free), (ii) immobilization on the biomolecular scaffold, and (iii) compartmentalization. In particular, a detailed overview is correlated to the natural counterpart with advances made in the field. We have then discussed the beneficial account of coassembly of multienzymes and provided a synopsis of the essential parameters in the rational coimmobilization design.

Knowledge, attitudes and practice preference regarding drug prescriptions of resident dental doctors: A quantitative study.

BACKGROUND: There has been an increasing need to examine undergraduate dental education in prescribing to confirm whether it is establishing the goal of producing safe and rational prescribers. Prescribing by dental doctors after graduation is influenced by a variety of factors: the different teaching approaches of the professors at the clinics and in the pharmacology course, fellow colleagues and even the information provided by the pharmaceutical industry. OBJECTIVE: The aim of this study was to assess the prescription knowledge, attitude, preference and common errors made by junior resident dental doctors in the dental department of two medical colleges and hospitals in India. METHODS: Dental graduates (n = 70) who served as junior resident doctors at dental departments and graduated from various dental colleges in India, participated in the study. A survey was conducted among 70 junior resident dental doctors in the dental department, applying a previously validated questionnaire consisting of open-ended questions. The knowledge, attitude and practice preference of resident doctors regarding drug prescription and common errors made by them were analyzed and reviewed. RESULTS: The most frequent response to each question was considered the most significant. The most common reason for prescribing medication was infection (n = 33, 47.14%) and the most widely prescribed antibiotic was amoxicillin (n = 29, 41.42%). The most frequent error reported by resident doctors was lack of knowledge about drug posology (n = 31, 44.28%). Maximum number of junior doctors acquired the information for prescribing drugs from their faculty (n = 33, 47.14%). Around 45.71% dental doctors had no knowledge about the WHO Guide to Good Prescribing. About 50- 60% doctors had knowledge about the dose, frequency, duration and route of administration of the drug prescribed. While prescribing drugs to the patient, 25.71% doctors explain the disease and treatment properly to the patient; 24.28% doctors do not criticize other doctors, and 10% doctors address the patient by name. CONCLUSION: The results of this study indicate that resident dental doctors' knowledge, attitudes and behavior about the drug prescription need to be improved. More studies are needed to determine whether this issue affects the quality of patient care and the effectiveness and safety of treatments.

Cyclophilin D knockout protects the mouse kidney against cyclosporin A-induced oxidative stress.

Mitochondrial dysfunction and oxidative stress have been implicated in cyclosporin A (CsA)-induced nephrotoxicity. CsA interacts with cyclophilin D (CypD), an essential component of the mitochondrial permeability transition pore and regulator of cell death processes. Controversial reports have suggested that CypD deletion may or may not protect cells against oxidative stress-induced cell death. In the present study, we treated wild-type (WT) mice and mice lacking CypD [peptidylprolyl isomerase F knockout (Ppif-/-) mice] with CsA to test the role and contribution of CypD to the widely described CsA-induced renal toxicity and oxidative stress. Our results showed an increase in the levels of several known uremic toxins as well as the oxidative stress markers PGF2α and 8-isoprostane in CsA-treated WT animals but not in Ppif-/- animals. Similarly, a decline in S-adenosylmethionine and the resulting methylation potential indicative of DNA hypomethylation were observed only in CsA-treated WT mice. This confirms previous reports of the protective effects of CypD deletion on the mouse kidney mediated through a stronger resistance of these animals to oxidative stress and DNA methylation damage. However, a negative effect of CsA on the glycolysis and overall energy metabolism in Ppif-/- mice also indicated that additional, CypD-parallel pathways are involved in the toxic effects of CsA on the kidney. In summary, CsA-mediated induction of oxidative stress is associated with CypD, with CypD deletion providing a protective effect, whereas the reduction of energy production observed upon CsA exposure did not depend on the animals' CypD status.

A comparative study to determine strength of autopolymerizing acrylic resin and autopolymerizing composite resin influenced by temperature during polymerization: An In Vitro study.

AIM: Temporary coverage of a prepared tooth is an important step during various stages of the fixed dental prosthesis. Provisional restorations should satisfy proper mechanical requirements to resist functional and nonfunctional loads. A few studies are carried out regarding the comparison of the effect of curing environment, air and water, on mechanical properties of autopolymerizing acrylic and composite resin. Hence, the aim of this study was to compare the transverse strength of autopolymerizing acrylic resin and autopolymerizing composite resin as influenced by the temperature of air and water during polymerization. MATERIALS AND METHODS: Samples of autopolymerizing acrylic resin and composite resin were prepared by mixing as per manufacturer's instructions and were placed in a preformed stainless steel mold. The mold containing the material was placed under different controlled conditions of water temperature and air at room temperature. Polymerized samples were then tested for transverse strength using an Instron universal testing machine. RESULTS: Alteration of curing condition during polymerization revealed a significant effect on the transverse strength. The transverse strength of acrylic resin specimens cured at 60°C and composite resin specimens cured at 80°C was highest. Polymerizing the resin in cold water at 10°C reduced the mechanical strength. CONCLUSIONS: Polymerization of the resin in hot water greatly increased its mechanical properties. The method of placing resin restoration in hot water during polymerization may be useful for improving the mechanical requirements and obtaining long-lasting performance.

Sturge Weber Syndrome: An Unusual Case with Multisystem Manifestations.

BACKGROUND: Sturge-Weber syndrome (SWS) is a rare congenital neurocutaneous disorder. It is characterized by the presence of facial port wine stains, neurological abnormalities like seizures and mental retardation, ocular disorders, oral involvement and leptomeningeal angiomas. CASE REPORT: A 13-year-old boy presented with the chief complaint of swollen, bleeding gums and deposits on the teeth. Detailed medical and dental history, clinical examination and investigations confirmed the diagnosis of Sturge-Weber syndrome. The treatment comprised of a thorough plaque control regimen to reduce the gingival enlargement, and it included oral hygiene instructions, thorough scaling, root planing at regular intervals and plaque index scoring which motivated the patient at each visit. CONCLUSION: This case illustrates that early intervention in a patient with Sturge-Weber syndrome is quintessential because of its associated gingival vascular features and their complicating manifestations. Furthermore, the need for periodic oral examinations and maintenance of good oral hygiene to prevent any complications from the oral vascular lesions has been highlighted.

Bienzymatic Sequential Reaction on Microgel Particles and Their Cofactor Dependent Applications.

We report, the preparation and characterization of bioconjugates, wherein enzymes pyruvate kinase (Pk) and l-lactic dehydrogenase (Ldh) were covalently bound to poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgel support using glutaraldehyde (GA) as the cross-linker. The effects of different arrangements of enzymes on the microgels were investigated for the enzymatic behavior and to obtain maximum Pk-Ldh sequential reaction. The dual enzyme bioconjugates prepared by simultaneous addition of both the enzymes immobilized on the same microgel particles (PL), and PiLi, that is, dual enzyme bioconjugate obtained by combining single-enzyme bioconjugates (immobilized pyruvate kinase (Pi) and immobilized lactate dehydrogenase (Li)), were used to study the effect of the assembly of dual enzymes systems on the microgels. The kinetic parameters (Km, kcat), reaction parameters (temperature, pH), stability (thermal and storage), and cofactor dependent applications were studied for the dual enzymes conjugates. The kinetic results indicated an improved turn over number (kcat) for PL, while the kcat and catalytic efficiency was significantly decreased in case of PiLi. For cofactor dependent application, in which the ability of ADP monitoring and ATP synthesis by the conjugates were studied, the activity of PL was found to be nearly 2-fold better than that of PiLi. These results indicated that the influence of spacing between the enzymes is an important factor in optimization of multienzyme immobilization on the support.

Smart core-shell microgel support for acetyl coenzyme A synthetase: a step toward efficient synthesis of polyketide-based drugs.

The flexibility in tuning the structure and charge properties of PNIPAm microgels during their synthesis makes them a suitable choice for various biological applications. Two-step free radical polymerization, a common method employed for synthesis of core-shell microgel has been well adopted to obtain cationic poly(N-isopropylacrylamide-aminoethyl methacrylate) (PNIPAm-AEMA) shell and PNIPAm core. Scanning electron microscopy (SEM), dynamic light scattering (DLS), zeta potential, and ninhydrin assay suggests nearly monodispersed particles of cationic nature. Amino groups on the microgel provides suitable attachment point for covalent immobilization of acetyl coenzyme A synthetase (Acs) via 1-ethyl-3-(3-N,N- dimethylaminopropyl) carbodiimide (EDC) chemistry. On immobilization, 61.55% of initial activity of Acs has been retained, while Michaelis-Menten kinetics of the immobilized Acs indicates identical K(m) (Michaelis constant) but decrease in the V(max) (maximum substrate conversion rate) compared to free enzyme. Immobilized Acs shows an improvement in activity at wide temperature and pH range and also demonstrates good thermal, storage, and operational stability. The Acs-microgel bioconjugate has been successfully reused for four consecutive operation cycles with more than 50% initial activity.

Functional polyelectrolyte multilayer membranes for water purification applications.

A diverse set of supported multilayer assemblies with controllable surface charge, hydrophilicity, and permeability to water and solute was fabricated by pressure driven permeation of poly(sodium 4-styrenesulfonate) (PSS) and poly(diallyldimethylammonium chloride) (PDDA) solution through poly(ethylene terephthalate) (PET) track-etched membranes. The polyelectrolyte multilayer fabrication was confirmed by means of FTIR, SEM, AFM, ellipsometry, zetapotential, and contact angle characterization. The prepared membranes were characterized in terms of their pure water permeability, flux recovery, and resistance to organic and biofouling properties. The antifouling behavior of the membranes was assessed in terms of protein adsorption and antibacterial behavior. Finally, the membranes were tested for rejection of selected water soluble dyes to establish their usefulness for organic contaminant removal from water. The membranes were highly selective and capable of nearly complete rejection of congo red with sufficiently high fluxes. The feasibility of regenerating the prepared membranes fouled by protein was also demonstrated and good flux recovery was obtained. In summary, the multilayer approach to surface and pore modification was shown to enable the design of membranes with the unique combination of desirable separation characteristics, regenerability of the separation layer, and antifouling behavior.

Antifouling and antibiofouling pH responsive block copolymer based membranes by selective surface modification.

Antifouling and antibacterial membranes are prepared by selective surface modification of pH responsive polystyrene-b-poly(4-vinylpyridine) (PS-P4VP) diblock copolymers by quaternization and zwitterionization reactions on a P4VP moiety. Nanoporous membranes based on the self-assembly of 2-(4'-hydroxybenzeneazo) benzoic acid (HABA)-PS-P4VP supramolecular complexes and nonsolvent induced phase separation are first prepared and the surfaces are functionalized by crosslinking with diiodobutane vapors and reacting with propane sultone vapors at moderate temperature and under vacuum conditions. Selective functionalization of surfaces is carried out to enhance the antifouling and antibiofouling properties of the membrane and to retain its pH switching behavior. The membranes are thoroughly characterized by various instrumental techniques such as Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, quartz crystal microbalance, contact angle, etc. Antifouling and antibacterial properties are proven by analyzing the adsorption of bovine serum albumin protein and bacterial cell attachment and killing efficiency. The actual membrane performance is assessed in terms of water flux under different pressures and pHs and Congo red dye rejection efficiency.

Evaluation of the clinical efficacy of glass fiber reinforced composite resin as a space maintainer and its comparison with the conventional band and loop space maintainer. An in vivo study.

AIM: Premature primary tooth loss may result in deleterious changes in dental arch integrity like space loss, crowding and midline shift, compromising the eruption of succedaneous teeth and altering the development of normal occlusion. The most confident way to cope with these problems is through the use of space maintainers. Today is an era of bondontics, which gives us a chance to test challenging materials like glass fiber reinforced composite resin (GFRCR) as a space maintainer. The objective of this study was to assess the clinical efficacy of GFRCR as a space maintainer and to compare it with the conventional Band and Loop space maintainer. METHODS: Twenty children aged 4-9 years requiring bilateral space maintainers for premature loss of primary first molar or primary second molar were selected for the study. GFRCR and Band and Loop space maintainers were applied bilaterally and their retention was evaluated at 1, 3 and 5 months interval. RESULTS: In comparison to band and loop space maintainers, the GFRCR space maintainers showed a higher success but the difference was statistically non significant. CONCLUSION: This study showed that GFRCR space maintainer can be a new alternative to the conventional space maintainer for short-term space maintenance for primary tooth loss.