High-purity 1,2-dimyristoyl-sn-glycero-3-phosphocholine: synthesis and emulsifying performance evaluation.

Introduction: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) is a promising emulsifier for bioactive delivery systems, but its industrial applications are limited by the lack of cost-effective and scalable synthetic routes. The purpose of this study was to economically produce high-purity DMPC by replacing commonly used column chromatography methods and to evaluate the emulsifying performance. Methods: DMPC was synthesized from sn-glycero-3-phosphocholine using Steglich esterification followed by sequential recrystallization from ethyl acetate and acetone. The structure of DMPC was identified and its purity was confirmed using various spectroscopy and chromatography techniques. The emulsifying performance was evaluated by examining the effects of storage on the properties of o/w emulsions prepared using soybean oil with (i) soy PC, (ii) soy PC + DMPC (1:1, w/w), and (iii) DMPC as emulsifiers. Results: The chemical impurities formed during the synthesis of DMPC was removed, and its final purity was 96%, and the melt transition temperature was 37.6°C. No visible difference between the three emulsions (soy PC, soy PC+DMPC, and DMPC) was observed during two-week storage, and the DMPC-based emulsion was more stable than soy PC emulsion, showing smaller particle size distribution during 6 months. Discussion: The highly pure DMPC was synthesized by an economical method, and DMPC-based emulsions demonstrated physicochemical stable, highlighting its potential for food and pharmaceutical industry-related applications. Our findings suggest that DMPC holds promise as an emulsifier with broad applications in the food industry.

Assessing the developmental competence of oocytes matured following rescue in vitro maturation: a systematic review and meta-analysis.

PURPOSE: To assess the developmental competence of oocytes matured following rescue in vitro maturation (IVM). METHODS: PubMed, EmBASE, and SCOPUS were systematically searched for peer-reviewed original papers using relevant keywords and Medical Subject Heading terms. Study quality was assessed using the Newcastle-Ottawa Scale. Odds ratios with a 95% confidence interval were calculated by applying a random effects model. The primary outcomes were fertilization and blastulation rates. Secondary outcomes included abnormal fertilization, cleavage, euploidy, clinical pregnancy, and live-birth rates. RESULT: Twenty-four studies were included in the meta-analysis. The oocytes matured following rescue IVM showed significantly reduced fertilization, cleavage, blastulation, and clinical pregnancy rates compared to sibling in vivo-matured oocytes. No significant differences were found for the euploidy and live-birth rates in euploid blastocyst transfer. In poor responders, a reduced fertilization rate was observed using in vitro-matured GV but not with in vitro-matured MI. A reduced cleavage rate in MI matured overnight compared to < 6 incubation hours was found. CONCLUSION: Our results showed compromised developmental competence in oocytes matured following rescue IVM. However, in poor responders, rescue IVM could maximize the efficiency of the treatment. Notably, our data suggests using in vitro MI matured within 6 incubation hours. CLINICAL TRIAL REGISTRATION NUMBER: CRD42023467232.

Simulating the Entire Clinical Process for an Implant-Supported Fixed Prosthesis: In Vitro Study on the Vertical Implications of Implant-Abutment Connections and Rotational Freedom.

OBJECTIVES: The aim of this in vitro study was to investigate whether and to what extent different scenarios of rotational freedom in different IAC designs affect the vertical dimension of a three-part fixed partial denture (FPD). At the same time, the experimental setup should simulate all clinical and laboratory steps of the implementation of such an FPD as accurately as possible. MATERIAL AND METHODS: Twenty identical pairs of jaw models were fabricated from aluminum, each lower-jaw model holding two implants with conical or flat IACs. Three impressions of each model were taken to fabricate stone casts and three-unit FPDs. Three assembly scenarios were compared for the vertical position stability they offered for these FPDs, differing by how the sequential implant components (impression posts > laboratory analogs > abutments 1 > abutments 2) were aligned with the positional index of the IAC. In this way, a total of 60 stone casts and FPDs were fabricated and statistically analyzed for changes in vertical dimension (p < 0.05). RESULTS: Regardless of whether a conical/flat IAC was used (p > 0.05), significantly greater mean changes in vertical dimension were consistently (all comparisons p < 0.0001) found in a "worst-case scenario" of component alignment alternating between the left- and right-limit stop of the positional index (0.286/0.350 mm) than in a "random scenario" of 10 dentists and 10 technicians with varying levels of experience freely selecting the alignment (0.003/0.014 mm) or in a "best-case scenario" of all components being aligned with the right-limit stop (-0.019/0.005 mm). CONCLUSIONS: The likelihood of integrating a superstructure correctly in terms of vertical dimension appears to vary considerably more with assembly strategies than with IAC designs. Specifically, our findings warrant a recommendation that all implant components should be aligned with the right-limit stop of the positioning index.

NPPC and AREG supplementation in IVM systems alter mRNA translation and decay programs-related gene expression in bovine COC.

During oocyte meiosis resumption, a coordinated program of transcript translation and decay machinery promotes a remodeling of mRNA stores, which determines the success of the acquisition of competence and early embryo development. We investigated levels of two genes related to mRNA translation (CPEB1 and CPEB4) and two related to mRNA degradation (CNOT7 and ZFP36L2) machinery and found ZFP36L2 downregulated in in vitro-matured bovine oocytes compared to in vivo counterparts. Thereafter, we tested the effects of a pre-IVM step with NPPC and a modified IVM with AREG on the modulation of members of mRNA translation and degradation pathways in cumulus cells and oocytes. Our data showed a massive upregulation of genes associated with translational and decay processes in cumulus cells, promoted by NPPC and AREG supplementation, up to 9h of IVM. The oocytes were less affected by NPPC and AREG, and even though ZFP36L2 transcript and protein levels were downregulated at 9 and 19h of IVM, only one (KDM4C) from the ten target genes evaluated was differently expressed in these treatments. These data suggest that cumulus cells are more prone to respond to NPPC and AREG supplementation in vitro, regarding translational and mRNA decay programs. Given the important nursing role of these cells, further studies could contribute to a better understanding of the impact of these modulators in maternal mRNA modulation and improve IVM outcomes.

Botrytis gray mold of Lilium in Bangladesh: Diagnosis, basic study and control.

Bangladesh Agricultural Research Institute (BARI) released two beautiful Lilium varieties in 2020. In the same year the farmers in Gazipur district reported a set of disease symptoms on these flowers and alerted the Plant Pathology Division of BARI. Subsequent investigation confirmed the symptoms as Botrytis gray mold (BGM), caused by Botrytis cinerea. The pathogen identity was confirmed through ITS gene sequencing. A series of in vitro and in planta experiments conducted to understand the symptoms, the optimal growth condition for the pathogen, potential resistant Lilium genotypes, effective chemical treatments and potential of biological control agents to combat the disease. B. cinerea exhibited the highest growth in V8 media (88.55 mm) at pH6 (85.32 mm) and temperature between 20 and 25 °C (89.56 mm), and pH6 (85.32 mm). Screening revealed that five oriental-originated genotypes provided lower disease incidence (31.66-41.66 %), and were categorized as moderately resistant to resistant in disease reaction. Six fungicides successfully reduced mycelial growth in vitro. Moreover, Ipridione provided the lowest % disease incidence (27.11) and % disease severity (5.33) in the in planta nethouse experiment. Therefore, this fungicide was recommended to the farmers initially. Additionally, two fungal biocontrol agents Epicoccum nigrum EJS002 and Trichoderma ThC003, demonstrated effectiveness in reducing leaf lesion size over control in a detach leaf assessment technique. In conclusion, this study presents BGM of Lilium as a farmers issue for the first time in Bangladesh. It also provides valuable insights into its management, recommending specific chemical fungicides for farmers to use, and two fungal antagonists (E. nigrum EJS002 and Trichoderma ThC003) as potential disease control agent.

Adaptation of an in-vitro digestion model with different zinc-supplementation strategies on nutrient degradation of piglets.

In-vitro studies are widely used in nutrition research. Two major challenges using in-vitro models in animal nutrition research are the individual adaptation of in-vitro digestion models to varying physiological conditions and small digesta output limiting sample material for further analysis. Since several years, the use of zinc in animal production has been legally reduced to control zinc emissions. Earlier, zinc doses around 3000 mg/kg diet were used to prevent post-weaning diarrhea and promote growth in weaning piglets. The first aim of this study was to adapt an in-vitro digestion system for piglets with increased sample output. The second aim was to study the effect of a titanium-bound zinc source at legal dietary inclusion levels on nutrient degradation in an in-vitro digestion model. The experiment was conducted in a 2x2 factorial design incubating 2 different feeds (1. control feed: a commercial piglet diet containing 75 mg zinc per kg diet and 2. treatment feed: control feed with 50 mg of a titanium-bound zinc oxide) in in-situ digestion bags in the Ankom Daisy® incubator with or without digestive enzymes (pepsin, pancreatic enzymes and bile salts). Residuals of incubated feed were analyzed for crude ash, crude protein and starch. The addition of pepsin, pancreatic enzymes and bile salts significantly increased organic matter, crude protein and starch degradation from the digested feed, therefore making the distinction of nutrient disappearance due to enzyme activity versus due to dissolution possible. In conclusion we established an in-vitro digestion model to evaluate the effect of addition of a new zinc source on the enzymatic digestion in piglets. However, addition of the new zinc source did not significantly improve nutrient degradation in the in-vitro digestion model.

First-trimester ultrasound diagnosis and risk factor analysis of cesarean scar pregnancy after in vitro fertilization-embryo transfer.

Background: Cesarean scar pregnancy (CSP) is one of the rarest ectopic pregnancies which may be associated with life-threatening complications. Owing to the rarity of CSP, little is known about it. This study aimed to evaluate the value of the first-trimester transvaginal sonography (TVS) diagnosis and the risk factors of CSP after in vitro fertilization-embryo transfer (IVF-ET). Methods: This was a retrospective study of women undergoing IVF-ET between January 2013 and December 2018. Women who were diagnosed with a CSP using TVS and confirmed by surgery and histological examination were included. The clinical data and ultrasound findings were collected and analyzed. Univariate and multivariate logistic regression analyses were performed for evaluation of possible influence factors. Diagnostic parameters including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TVS were calculated for the diagnosis of CSP. Results: Overall, 75,438 consecutive women who underwent IVF-ET had received TVS during this period. Of these, 4,817 women (6.4%) had a history of cesarean section and 83 cases were found to have a CSP. Due to the absence of histological data, 19 cases treated conservatively were excluded. Finally, 64 cases were included, among whom 63 cases were correctly diagnosed [including 17 cases of heterotopic CSP (HCSP)] and 1 case was missed using TVS. Another 1 case of inevitable miscarriage was misdiagnosed as a CSP. The maternal age at the initial scan [34.0 (range, 26.0-44.0) years], the infertility duration [4.0 (range, 1-12) years], and the initial diagnostic time after ET [27 (range, 20-50) days] were recorded. A gestational sac (GS) was observed in all 63 cases during ultrasound examinations, including 28 with fetal pole, 25 with a yolk sac only, and 10 with an empty sac. The sensitivity and specificity of first-trimester TVS in diagnosing CSP were 98.44% and 99.98%, respectively; the PPV and NPV were 98.44% and 99.98%, respectively. Multivariate logistic regression analysis showed thinner endometrial thickness (ET) on transfer day [adjusted odds ratio (aOR): 0.83; 95% confidence interval (CI): 0.76-0.93, P<0.001] and multiple ET (aOR 53.60, 95% CI: 5.31-1,736.00, P=0.008) were independent risk factors for CSP and HCSP, respectively. Conclusions: First-trimester TVS performed by an experienced sonographer has a high sensitivity for making the correct diagnosis of CSP after IVF-ET, which is helpful for clinical intervention and avoiding severe complications. For patients with a history of cesarean section, thinner ET on the transfer day and bigger body mass index (BMI) seem to be risk factors for CSP; single blastocyst transfer should be recommended to decrease the possibility of HCSP. The clinical significance of this study still needs to be considered.

Zinc oxide nanoparticles loaded with linalool as a potential control agent of malaria infection.

This research aimed to produce and analyze zinc oxide nanoparticles (ZNPs) loaded with linalool (LZNPs), and to evaluate their in vitro and in vivo efficacy through targeting the inflammation and oxidative stress. LZNPs were synthesized using an ethanolic solution of polyvinyl alcohol. The Malstat technique was used to evaluate the effectiveness of LZNPs against both sensitive and resistant strains of Plasmosium falciparum. In vivo effects of ZNPs and LZNPs on parasite growth suppression, survival rate, oxidative stress markers, antioxidant genes, and gene and protein levels of inflammatory cytokines were evaluated by Real-time PCR and Western blot techniques. The results indicated that LZNPs demonstrated noteworthy (P < 0.001) antiplasmodial activity against both susceptible and resistant strains of P. falciparum. P. berghei NK65 strain-infected mice treated with the ZNPs and LZNPs at doses of 5-15 mg/kg notably (p < 0.001) increased the survival rates and parasite growth suppression. LZNPs at 5-15 mg/kg demonstrated a significant (p < 0.001) decrease in oxidative stress markers, increased the expression level of antioxidant genes, and reduced the gene and protein expression level of inflammatory cytokines. The current experimental study demonstrated the potent in vitro antiplasmodial activity of LZNPs against chloroquine-resistant and sensitive strains of P. falciparum compared to ZNPs alone. Additionally, the study identified the potential benefits of this nanocomposite in suppressing the parasite and extending the survival rate in mice infected with P. berghei by targeting inflammation and oxidative stress. It also showed minimal toxicity in liver and kidney function in healthy mice. Nevertheless, further research is essential to elucidate the comprehensive mechanisms and practical effectiveness of LZNPs.

Effects of debranched starch on physicochemical properties and in vitro digestibility of flat rice noodles.

Aiming to develop flat rice noodles with both desirable textural quality and lower starch digestibility, we investigated the effect of adding indica rice debranched starch (RDBS) on the quality of flat rice noodles. In this study, adding RDBS to flat rice noodles enhanced their mechanical properties. Cooking characteristic analysis showed that incorporating RDBS into dried flat rice noodles increased the rehydration ratio by 16.1 % and reduced rehydration time by 26.5 %. Scanning electron microscopy (SEM) revealed the presence of microparticles formed through the self-assembly of RDBS within the network of flat rice noodles. X-ray diffraction (XRD) analysis demonstrated that the addition of RDBS elevated the crystallinity of the flat rice noodles, rising from 9.59 % to 22.57 %. In addition, the in vitro simulated digestion test suggested the addition of RDBS led to a threefold increase in the content of slowly digestible starch (SDS) and a ninefold increase in resistant starch (RS) content in flat rice noodles. This study found that adding RDBS into flat rice noodles can effectively reduce their digestion rate and improve their eating quality. It could be a promising approach for creating functional rice noodles aimed at alleviating public health concerns such as diabetes and obesity.

Posaconazole-hemp seed oil loaded nanomicelles for invasive fungal disease.

Invasive fungal infections (IFI) pose a significant health burden, leading to high morbidity, mortality, and treatment costs. This study aims to develop and characterize nanomicelles for the codelivery of posaconazole and hemp seed oil for IFI via the oral route. The nanomicelles were prepared using a nanoprecipitation method and optimized through the Box Behnken design. The optimized nanomicelles resulted in satisfactory results for zeta potential, size, PDI, entrapment efficiency, TEM, and stability studies. FTIR and DSC results confirm the compatibility and amorphous state of the prepared nanomicelles. Confocal laser scanning microscopy showed that the optimized nanomicelles penetrated the tissue more deeply (44.9µm) than the suspension (25µm). The drug-loaded nanomicelles exhibited sustained cumulative drug release of 95.48 ± 3.27% for 24 h. The nanomicelles showed significant inhibition against Aspergillus niger and Candida albicans (22.4 ± 0.21 and 32.2 ± 0.46 mm, respectively). The pharmacokinetic study on Wistar rats exhibited a 1.8-fold increase in relative bioavailability for the nanomicelles compared to the suspension. These results confirm their therapeutic efficacy and lay the groundwork for future research and clinical applications, providing a promising synergistic antifungal nanomicelles approach for treating IFIs.

Insight into the mechanism of digestibility inhibition by interaction between corn starch with different gelatinization degree and water extractable arabinoxylan.

On the basis of revealing the interaction mechanism between corn starch (CS) and water-extractable arabinoxylan (WEAX) with high/low molecular weight (H-WEAX, L-WEAX), the degree of gelatinization (DG) on structural behaviors and in vitro digestibility of CS-WEAX complexes (CS/H, CS/L) was evaluated. With the increased DG from 50 % to 95 %, the water adsorption capacity of CS/L was increased 64 %, 58 %, 47 %, which were higher than that of CS/H (39 %, 54 %, 33 %). The gelatinization of starch was inhibited by WEAX, resulting in the enhancement of crystallinity, short-range ordered structure and molecular size of CS-WEAX complexes. Stronger interaction was detected in CS/L than with CS/H as proved by the increased hydrogen bonds and electrostatic force. Complexes exhibited higher resistant starch content (RS) at diverse DG, especially for CS/L. Notability, RS content of samples with 50 % DG were increased from 27.72 % to 32.89 % (CS/H), 36.96 % (CS/L). Except for the reduction of gelatinization degree by adding WEAX, the other possible mechanisms of retarding digestibility were explained as the small steric hindrance of L-WEAX promoted encapsulation of starch granules, limiting enzyme accessibility. Additionally, the fragmentation of CS granules with high DG promoted the movement of H-WEAX, reducing the difference in digestibility compared to CS/L.

Preparation, characterization, and liver targeting evaluation of a novel sustained-release brucine self-assembled micelle mediated by glycyrrhetinic acid.

Background: Cancer is a serious threat to human life, health and social development. In recent years, nanomicelles, as an emerging drug carrier material, have gradually entered people's field of vision because of their advantages of improving bioavailability, maintaining drug levels, reducing systemic side effects and increasing drug accumulation at target sites. Methods: In this study, B-GPSG nano-micelles were prepared by film dispersion hydration method using brucine as model drug and glycyrrhetinic acid-polyethylene glycol-3-methylene glycol-dithiodipropionic acid-glycerol monostearate polymer as nano-carrier. The preparation process, characterization, drug release in vitro, pharmacokinetics and liver targeting were investigated. Results: The results showed that the range of particle size, polydispersion index and Zeta potential were 102.7 ± 1.09 nm, 0.201 ± 0.02 and -24.5 ± 0.19 mV respectively. The entrapment efficiency and drug loading were 83.79 ± 2.13% and 12.56 ± 0.09%, respectively. The drug release experiments in vitro and pharmacokinetic experiments showed that it had obvious sustained release effect. For pharmacokinetics study, it shows that both the B-GPSG solution group and the B-PSG solution group changed the metabolic kinetic parameters of brucine, but the B-GPSG solution group had a better effect. Compared with the B-PSG solution group, the drug was more prolonged in rats. The half-life in the body and the retention time in the body of B-GPSG are more helpful to improve the bioavailability of the drug and play a long-term effect. The tail vein injection results of mice indicate that B-GPSG can target and accumulate brucine in the liver without affecting other key organs. Cell uptake experiments and tissue distribution experiments in vivo show that glycyrrhetinic acid modified nano-micelles can increase the accumulation of brucine in hepatocytes, has a good liver targeting effect, and can be used as a new preparation for the treatment of liver cancer. Conclusion: The B-SPSG prepared in this experiment can provide a new treatment method and research idea for the treatment of liver cancer.

Applications and evolution of 3D cancer-immune cell models.

Understanding the highly complex tumor-immune landscape is an important goal for developing novel immune therapies for solid cancers. To this end, 3D cancer-immune models have emerged as patient-relevant in vitro tools for modeling the tumor-immune landscape and the cellular interactions within it. In this review, we provide an overview of the components and applications of 3D cancer-immune models and discuss their evolution from 2015 to 2023. Specifically, we observe trends in primary cell-sourced, T cell-based complex models used for therapy evaluation and biological discovery. Finally, we describe the challenges of implementing 3D cancer-immune models and the opportunities for maximizing their potential for deciphering the complex tumor-immune microenvironment and identifying novel, clinically relevant drug targets.

Association between subchorionic hematoma in the first trimester and outcomes of singleton pregnancies achieved through assisted reproductive technology: a systematic review and meta-analysis.

PURPOSE: To evaluate the association between first trimester (≤ 12 weeks gestation) subchorionic hemorrhage (SCH), and maternal and neonatal outcomes in women who conceived with the help of assisted reproductive technique (ART). METHODS: PubMed, Embase, Web of Science, and Scopus databases were searched for observational studies that specifically focused on women who achieved pregnancy via ART and investigated the relationship between early pregnancy (within 12 weeks of gestation) SCH and maternal and neonatal outcomes. Only studies with singleton pregnancies and reporting data on the comparator group (women without SCH) were included. Primary outcomes of interest included incidences of early (within 20 weeks of gestation) pregnancy loss, preterm delivery, caesarean section, and live birth rates. Pooled effect sizes were reported as odds ratio (OR) with 95% confidence intervals (CI). RESULTS: Nine studies were included. All studies had a cohort design. In all studies, the primary assisted reproduction technique used was in-vitro fertilization (IVF). Compared to pregnancies without SCH, women with diagnosed early pregnancy SCH have a similar risk of preterm birth (< 37 weeks) (OR 1.01, 95% CI 0.83, 1.22), low birth weight (< 2500 g) (OR 1.01, 95% CI 0.59, 1.73) and fetal growth restriction (OR 1.57, 95% CI 0.62, 4.02). The gestational age (in weeks) (weighted mean difference (WMD) - 0.06, 95% CI - 0.18, 0.06) and the birth weight (in grams) (WMD - 16.5, 95% CI - 62.9, 29.8) were also similar in the two groups. The odds of early pregnancy loss (OR 1.39, 95% CI 0.97, 2.01), live birth (OR 0.77, 95% CI 0.55, 1.08) and caesarean delivery (OR 0.97, 95% CI 0.81, 1.16) were statistically similar in both groups. The risk of maternal adverse outcomes such as gestational diabetes (OR 0.98, 95% CI 0.74, 1.29), hypertensive disorder (OR 0.95, 95% CI 0.63, 1.43), premature rupture of membranes (PROM) (OR 1.36, 95% CI 0.90, 2.05) and placental abruption (OR 2.44, 95% CI 0.57, 10.5) was also similar in both the groups. There was no evidence of publication bias. CONCLUSION: The findings suggest that SCH may not significantly increase the risk of adverse maternal and perinatal outcomes in pregnancies conceived through ART, particularly IVF. TRIAL REGISTRATION: PROSPERO registration number CRD42024533996.

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform.

Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5' and 3' untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5'UTRs, 10 3'UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.

In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.

Three nonconventional starch: Comparison of physicochemical properties and in vitro digestibility.

Extraction of starch from waste is also an effective way to recover resources and provide new sources of starch. In this study, starch was isolated from white kidney bean residue, chickpea residue, and tiger nut meal after protein or oil extraction, and the morphology of starch particles was observed to determine their physicochemical properties and in vitro digestibility. All these isolated starches had unique properties, among which white kidney bean starch (KBS) had a high amylose content (43.48%), and its structure was better ordered. Scanning electron microscopy revealed distinct granular morphologies for the three starches. KBS and chickpea starch (CHS) were medium-granular starches, whereas tiger nut starch was a small granular starch. Fourier transform infrared spectroscopy analysis confirmed the absence of significant differences in functional groups and chemical bonds among the three starch molecules. In vitro digestibility studies showed that CHS is more resistant to enzymatic degradation. Overall, these results will facilitate the development of products based on the separation of nonconventional starches from waste.

SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression.

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed "stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes" (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper's transparent peer review process is included in the supplemental information.

Morphotype-specific calcium signaling in human microglia.

BACKGROUND: Key functions of Ca2+ signaling in rodent microglia include monitoring the brain state as well as the surrounding neuronal activity and sensing the danger or damage in their vicinity. Microglial Ca2+ dyshomeostasis is a disease hallmark in many mouse models of neurological disorders but the Ca2+ signal properties of human microglia remain unknown. METHODS: We developed a novel genetically-encoded ratiometric Ca2+ indicator, targeting microglial cells in the freshly resected human tissue, organotypically cultured tissue slices and analyzed in situ ongoing Ca2+ signaling of decades-old microglia dwelling in their native microenvironment. RESULTS: The data revealed marked compartmentalization of Ca2+ signals, with signal properties differing across the compartments and resident morphotypes. The basal Ca2+ levels were low in ramified and high in ameboid microglia. The fraction of cells with ongoing Ca2+ signaling, the fraction and the amplitude of process Ca2+ signals and the duration of somatic Ca2+ signals decreased when moving from ramified via hypertrophic to ameboid microglia. In contrast, the size of active compartments, the fraction and amplitude of somatic Ca2+ signals and the duration of process Ca2+ signals increased along this pathway.

The impact of mitochondrial impairments on sperm function and male fertility: a systematic review.

BACKGROUND: Besides adenine triphosphate (ATP) production for sustaining motility, the mitochondria of sperm also host other critical cellular functions during germ cell development and fertilization including calcium homeostasis, generation of reactive oxygen species (ROS), apoptosis, and in some cases steroid hormone biosynthesis. Normal mitochondrial membrane potential with optimal mitochondrial performance is essential for sperm motility, capacitation, acrosome reaction, and DNA integrity. RESULTS: Defects in the sperm mitochondrial function can severely harm the fertility potential of males. The role of sperm mitochondria in fertilization and its final fate after fertilization is still controversial. Here, we review the current knowledge on human sperm mitochondria characteristics and their physiological and pathological conditions, paying special attention to improvements in assistant reproductive technology and available treatments to ameliorate male infertility. CONCLUSION: Although mitochondrial variants associated with male infertility have potential clinical use, research is limited. Further understanding is needed to determine how these characteristics lead to adverse pregnancy outcomes and affect male fertility potential.