Inhibition of the MLCK/MLC2 pathway protects against intestinal heat stroke-induced injury in rats.

Intestinal barrier dysfunction often exists in the heat stroke (HS) pathological process, which increases intestinal permeability and induces endotoxemia. The upregulation of MLCK is a crucial player affecting intestinal permeability. This study aimed to explore whether inhibiting myosin light chain kinase (MLCK) can improve HS-induced intestinal injury in rats. Twelve-week-old Wistar male rats were divided into three groups: the control group, the HS model group, and the treatment group [HS model + ML-7 (MLCK inhibitor)]. HS impaired the tight junctions in the rat gut and increased permeability. Additionally, increased inflammatory factors in serum, activation of apoptosis, and downregulation of tight junction proteins were observed in intestinal cells. ML-7 significantly inhibited the MLCK/p-MLC2 signaling pathway, increased the expression of tight junction proteins, reduced intestinal permeability, reduced apoptosis and alleviated the intestinal damage caused by HS. ML-7 inhibited HS-induced apoptosis of intestinal epithelial cells by regulating the ERK/p38/HSP70 axis. Furthermore, inhibition of MLCK upregulated HSP70 expression through activation of the ERK pathway and inhibited cell apoptosis by abolishing the p38 MAPK pathway. In conclusion, inhibiting the MLCK/p-MLC2 signaling pathway reduces HS-induced intestinal permeability and protects the intestinal mucosal barrier.

Correction: Fast label-free recognition of NRBCs by deep-learning visual object detection and single-cell Raman spectroscopy.

Correction for 'Fast label-free recognition of NRBCs by deep-learning visual object detection and single-cell Raman spectroscopy' by Teng Fang et al., Analyst, 2022, https://doi.org/10.1039/D2AN00024E.

Fast label-free recognition of NRBCs by deep-learning visual object detection and single-cell Raman spectroscopy.

Nucleated red blood cells (NRBCs) as a type of rare cell present in an adult's peripheral blood is a concern in hematology, intensive care medicine and prenatal diagnostics. However, it is labor-intensive to screen such rare cells from real complex cell mixtures especially in a label-free way. Herein, we report a new label-free method that incorporates image recognition and Raman spectroscopy for fast recognition of the rare cells in blood. First, we identified unlabeled NRBCs based on both Raman signals of hemoglobin and nucleated morphology, and recorded their microscopic image characteristics which were different enough from other blood cells in unlabeled morphology. Then, two deep-learning algorithms of visual object detection, Faster RCNN and YOLOv3, were investigated for cell morphological recognition on a low-cost computer configuration, and YOLOv3 was demonstrated to be more competent for real-time detection despite slightly lower precision. Finally, several NRBCs were successfully found in maternal blood using this method, which verified the methodological feasibility. Thus, we believe such a labor-saving approach might inspire a new idea for detecting rare cells from complex cell mixtures in a label-free and computer-assisted way.

Anti-Cancer Drug Sensitivity Assay with Quantitative Heterogeneity Testing Using Single-Cell Raman Spectroscopy.

A novel anti-cancer drug sensitivity testing (DST) approach was developed based on in vitro single-cell Raman spectrum intensity (RSI). Generally, the intensity of Raman spectra (RS) for a single living cell treated with drugs positively relates to the sensitivity of the cells to the drugs. In this study, five cancer cell lines (BGC 823, SGC 7901, MGC 803, AGS, and NCI-N87) were exposed to three cytotoxic compounds or to combinations of these compounds, and then they were evaluated for their responses with RSI. The results of RSI were consistent with conventional DST methods. The parametric correlation coefficient for the RSI and Methylthiazolyl tetrazolium assay (MTT) was 0.8558 ± 0.0850, and the coefficient of determination was calculated as R² = 0.9529 ± 0.0355 for fitting the dose⁻response curve. Moreover, RSI data for NCI-N87 cells treated by trastuzumab, everolimus (cytostatic), and these drugs in combination demonstrated that the RSI method was suitable for testing the sensitivity of cytostatic drugs. Furthermore, a heterogeneity coefficient H was introduced for quantitative characterization of the heterogeneity of cancer cells treated by drugs. The largest possible variance between RSs of cancer cells were quantitatively obtained using eigenvalues of principal component analysis (PCA). The ratio of H between resistant cells and sensitive cells was greater than 1.5, which suggested the H-value was effective to describe the heterogeneity of cancer cells. Briefly, the RSI method might be a powerful tool for simple and rapid detection of the sensitivity of tumor cells to anti-cancer drugs and the heterogeneity of their responses to these drugs.

3D genome of multiple myeloma reveals spatial genome disorganization associated with copy number variations.

The Hi-C method is widely used to study the functional roles of the three-dimensional (3D) architecture of genomes. Here, we integrate Hi-C, whole-genome sequencing (WGS) and RNA-seq to study the 3D genome architecture of multiple myeloma (MM) and how it associates with genomic variation and gene expression. Our results show that Hi-C interaction matrices are biased by copy number variations (CNVs) and can be used to detect CNVs. Also, combining Hi-C and WGS data can improve the detection of translocations. We find that CNV breakpoints significantly overlap with topologically associating domain (TAD) boundaries. Compared to normal B cells, the numbers of TADs increases by 25% in MM, the average size of TADs is smaller, and about 20% of genomic regions switch their chromatin A/B compartment types. In summary, we report a 3D genome interaction map of aneuploid MM cells and reveal the relationship among CNVs, translocations, 3D genome reorganization, and gene expression regulation.

Dynamic characterization of drug resistance and heterogeneity of the gastric cancer cell BGC823 using single-cell Raman spectroscopy.

Drug resistance and heterogeneous characteristics of human gastric carcinoma cells (BGC823) under the treatment of paclitaxel (PTX) were investigated using single-cell Raman spectroscopy (RS). RS of normal and drug-resistant BGC823 cells (DR-BGC823) were collected and analyzed using arithmetic, statistic and individual spectrum analysis. The dynamic effects of paclitaxel (PTX) in normal and DR-BGC823 cells were evaluated dynamically. The RS intensity changed with PTX over time and produced distinct different results for the two types of cells. The average RS intensities of the normal BGC823 cells initially decreased and then increased under PTX treatment after 24 hours. In contrast, upon exposure to PTX, the average intensity of the DR-BGC823 cells initially increased within 12 hours and then gradually decreased and approached a steady state. The temporal variation of the typical component in the cells was analyzed by comparing the ratios between Raman bands. More importantly, the heterogeneous characteristics of the BGC823 cells under PTX treatment were quantified and clustered using hierarchical trees combined with RS intensity changes. The 'outlier' cells related to drug resistance were discriminated. The heterogeneity of the normal BGC823 cells under drug treatment gradually appeared over time, and was evaluated with the eigenvalues of principal component analysis (PCA). Our study indicates that single-cell RS may be useful in systematically and dynamically characterizing the drug response of cancer cells at the single-cell level.

ULtiMATE system for rapid assembly of customized TAL effectors.

Engineered TAL-effector nucleases (TALENs) and TALE-based constructs have become powerful tools for eukaryotic genome editing. Although many methods have been reported, it remains a challenge for the assembly of designer-based TALE repeats in a fast, precise and cost-effective manner. We present an ULtiMATE (USER-based Ligation Mediated Assembly of TAL Effector) system for speedy and accurate assembly of customized TALE constructs. This method takes advantage of uracil-specific excision reagent (USER) to create multiple distinct sticky ends between any neighboring DNA fragments for specific ligation. With pre-assembled templates, multiple TALE DNA-binding domains could be efficiently assembled in order within hours with minimal manual operation. This system has been demonstrated to produce both functional TALENs for effective gene knockout and TALE-mediated gene-specific transcription activation (TALE-TA). The feature of both ease-of-operation and high efficiency of ULtiMATE system makes it not only an ideal method for biologic labs, but also an approach well suited for large-scale assembly of TALENs and any other TALE-based constructions.

Results of phase II levetiracetam trial following acute head injury in children at risk for posttraumatic epilepsy.

Posttraumatic seizures develop in up to 20% of children following severe traumatic brain injury (TBI). Children ages 6-17 years with one or more risk factors for the development of posttraumatic epilepsy, including presence of intracranial hemorrhage, depressed skull fracture, penetrating injury, or occurrence of posttraumatic seizure were recruited into this phase II study. Treatment subjects received levetiracetam 55 mg/kg/day, b.i.d., for 30 days, starting within 8 h postinjury. The recruitment goal was 20 treated patients. Twenty patients who presented within 8-24 h post-TBI and otherwise met eligibility criteria were recruited for observation. Follow-up was for 2 years. Forty-five patients screened within 8 h of head injury met eligibility criteria and 20 were recruited into the treatment arm. The most common risk factor present for pediatric inclusion following TBI was an immediate seizure. Medication compliance was 95%. No patients died; 19 of 20 treatment patients were retained and one observation patient was lost to follow-up. The most common severe adverse events in treatment subjects were headache, fatigue, drowsiness, and irritability. There was no higher incidence of infection, mood changes, or behavior problems among treatment subjects compared to observation subjects. Only 1 (2.5%) of 40 subjects developed posttraumatic epilepsy (defined as seizures >7 days after trauma). This study demonstrates the feasibility of a pediatric posttraumatic epilepsy prevention study in an at-risk traumatic brain injury population. Levetiracetam was safe and well tolerated in this population. This study sets the stage for implementation of a prospective study to prevent posttraumatic epilepsy in an at-risk population.

Quantitation of gamma-hydroxybutyric acid in dried blood spots: feasibility assessment for newborn screening of succinic semialdehyde dehydrogenase (SSADH) deficiency.

OBJECTIVE: SSADH deficiency, the most prevalent autosomal recessive disorder of GABA degradation, is characterized by elevated gamma-hydroxybutyric acid (GHB). Neurological outcomes may be improved with early intervention and anticipatory guidance. Morbidity has been compounded by complications, e.g. hypotonia, in undiagnosed infants with otherwise routine childhood illnesses. We report pilot methodology on the feasibility of newborn screening for SSADH deficiency. METHOD: Dried blood spot (DBS) cards from patients affected with SSADH deficiency were compared with 2831 archival DBS cards for gamma-hydroxybutyric acid content. Following extraction with methanol, GHB in DBS was separated and analyzed using ultra high-performance liquid chromatography tandem mass spectrometry. RESULTS: Methodology was validated to meet satisfactory accuracy and reproducibility criteria, including intra-day and inter-day validation. Archival refrigerated dried blood spot samples of babies, infants and children (N = 2831) were screened for GHB, yielding a mean +/- S.D. of 8 ± 5 nM (99.9%-tile 63 nM) (Min 0.0 Max 78 nM). The measured mean and median concentrations in blood spots derived from seven SSADH deficient patients were 1182 nM and 699 nM respectively (Min 124, Max 4851 nM). CONCLUSIONS: GHB concentration in all 2831 dried blood spot cards was well below the lowest concentration of affected children. These data provide proof-of-principle for screening methodology to detect SSADH deficiency with applicability to newborn screening and earlier diagnosis.

Partial Pyridoxine Responsiveness in PNPO Deficiency.

OBJECTIVE: Autosomal-recessive pyridox(am)ine phosphate oxidase (PNPO) deficiency causes pyridoxal-5-phosphate (PLP)-dependent epilepsy. We describe partial PNPO deficiency with a transient response to pyridoxine (B6). METHODS: CSF neurotransmitter metabolites, PLP, and amino acids were analyzed while the patient was receiving pyridoxine. PNPO gene sequencing was performed by standard techniques. RESULTS: A full-term 3,220 g male with refractory neonatal seizures became seizure free for 6 weeks on pyridoxine (B6). Breakthrough seizures followed. These stopped upon the first dose of PLP although episodes occurred as a dose became due. An unidentified peak was detected on the chromatographic system used to measure CSF PLP. PNPO gene sequencing identified a homozygous mutation in a highly conserved area in exon 3: c.352G>A p.G118R, predicting substitution of arginine for glycine. At age 28 months the child has hypotonia and developmental delay, both mild in severity. CONCLUSIONS: Transient pyridoxine responsiveness may be seen in partial PNPO deficiency. A CSF metabolite peak, likely pyridoxine phosphate, is identifiable in patients with PNPO deficiency who are taking supplemental pyridoxine. Partial B6 responsiveness is an indication for possible PNPO deficiency and trial of PLP.