[Protective effects of 2-methoxyestradiol against hypoxic pulmonary hypertension in neonatal rats]. / 2-ç”²æ°§åŸºé›ŒäºŒé†‡å¯¹æ–°ç”Ÿå¤§é¼ ç¼ºæ°§æ€§è‚ºåŠ¨è„‰é«˜åŽ‹çš„ä¿æŠ¤ä½œç”¨.

OBJECTIVES: To investigate the protective effects of 2-methoxyestradiol (2ME) against hypoxic pulmonary hypertension (HPH) in neonatal rats. METHODS: Ninety-six Wistar neonatal rats were randomly divided into a normoxia group, a hypoxia group, and a hypoxia + 2ME group, with each group further subdivided into 3-day, 7-day, 14-day, and 21-day subgroups, containing eight rats each. The hypoxia and hypoxia + 2ME groups received daily subcutaneous injections of saline and 2ME (240 µg/kg), respectively, while the normoxia group was raised in a normoxic environment with daily saline injections. Right ventricular systolic pressure (RVSP) was measured using the direct pressure method. Pulmonary vascular morphology was assessed using hematoxylin and eosin staining, with metrics including the percentage of medial thickness of small pulmonary arteries relative to the external diameter (MT%) and the cross-sectional area of the media of small pulmonary arteries relative to the total cross-sectional area (MA%). Immunohistochemistry was used to detect the expression levels of hypoxia-inducible factor-1α (HIF-1α) and proliferating cell nuclear antigen (PCNA) proteins, while real-time quantitative PCR was used to to assess HIF-1α and PCNA mRNA levels. RESULTS: Compared to the normoxia group, the hypoxia and hypoxia + 2ME groups showed increased RVSP and upregulated HIF-1α and PCNA protein and mRNA expression levels at 3, 7, 14, and 21 days after hypoxia (P<0.05). Furthermore, at 7, 14, and 21 days after hypoxia, the hypoxia group showed increased MT% and MA% (P<0.05). In comparison to the hypoxia group, the hypoxia + 2ME group exhibited reduced RVSP and downregulated HIF-1α and PCNA protein and mRNA expression levels, along with decreased MT% and MA% at 7, 14, and 21 days after hypoxia (P<0.05). CONCLUSIONS: 2ME may protect against HPH in neonatal rats by inhibiting the expression of HIF-1α and PCNA and reducing pulmonary vascular remodeling. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 757-764.

Using peptide barcodes for simultaneous profiling of protein expression from mRNA.

RATIONALE: mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression. METHODS: We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels. RESULTS: The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes. CONCLUSIONS: The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.

Protocol for in vivo elimination of avian auditory hair cells, multiplexed mRNA detection, immunohistochemistry, and S-phase labeling.

The avian inner ear can naturally regenerate sensory hair cells and is therefore an ideal candidate for investigating mechanisms leading to hair cell regeneration and functional recovery. Here, we present a surgical protocol for eliminating auditory hair cells via sisomicin injection into the lateral semicircular canal. We describe steps for multiplex mRNA detection in chicken basilar papilla and utricle sections. We then detail procedures for integrating immunohistochemistry for concurrent mRNA and protein visualization, complemented by S-phase labeling with EdU. For complete details on the use and execution of this protocol, please refer to Benkafadar et al., Benkafadar et al., Sato et al., Janesick et al., Scheibinger et al.1,2,3,4,5.

Liquid and Solid Hybridization Methods to Detect RNAs.

Northern blotting (NB) has been a long-standing method for RNA detection. However, its labor-intensive nature, reliance on high-quality RNA, and use of radioactivity have diminished its appeal over time. Nevertheless, the emergence of microRNAs (miRNAs) has reignited the demand for sensitive and quantitative NB techniques. We have recently developed cost-effective and rapid protocols for RNA detection using solid and liquid hybridization (LH) techniques which exhibit high sensitivity without the need for radioactive or specialized reagents like locked nucleic acid (LNA) probes. Our assays incorporate biotinylated probes and improved techniques for probe hybridization, transfer, cross-linking, and signal enhancement. We demonstrate that while NB is sensitive in detecting mRNAs and small RNAs, our LH protocol efficiently detects these as well as miRNAs at lower amounts of RNA, achieving higher sensitivity comparable to radiolabeled probes. Compared to NB, LH offers benefits of speed, sensitivity, and specificity in detecting mRNAs, small RNAs, and miRNAs.

Quantum Dots-Based Protocols for the Detection of RNAs.

RNA (ribonucleic acid) plays a crucial role in various cellular processes and is involved in the development and progression of several diseases. RNA molecules have gained considerable attention as potential biomarkers for various ailments, as they reflect the activity of genes in a particular cell or tissue. By measuring the levels of specific RNA molecules, such as messenger RNA (mRNA), noncoding RNAs, including microRNAs (miRNAs), and long noncoding RNAs (lncRNAs), researchers can infer the expression patterns of genes associated with a particular disease. Aberrant expression of specific miRNAs or lncRNAs has been associated with conditions such as cancer, cardiovascular diseases, neurodegenerative disorders, and more. Detection and quantification of these RNAs in biological samples, such as blood or tissue, can provide valuable diagnostic or prognostic information. Yet their analysis is a challenging endeavor due to their length, sequence similarity across family members, sensitivity to disintegration, and low quantity in total samples. New advances in nanophotonics have provided novel options for fabrication of quantum dots (QDs)-based biosensing devices capable of detecting a variety of disease-specific RNAs. Thus, we proposed and designed a nanophotonic method employing oligonucleotide-conjugated quantum dot nanoconjugates for the rapid and accurate detection of RNAs. Despite the abundance of other molecules in the sample, the approach delivers highly selective, precise identification of the target RNAs. The data also indicated the method's great practicality and simplicity in determining RNAs selectively. Overall, the approach enables the evaluation of RNA expression in relation to the initial onset and progression of a human health disorder.

Strand displacement-triggered FRET nanoprobe tracking TK1 mRNA in living cells for ratiometric fluorimetry of nucleic acid biomarker.

A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.

Integration of Hybridization Chain Reaction and Protein-Binding Amplification for Long-Term Imaging of Intracellular mRNA: Avoiding Signal Fluctuation.

Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.

Enhancing RNA detection and breast cancer subtyping with a universal 3D-hybridization chain reaction system.

Breast cancer, a globally prevalent malignancy, is characterized by pronounced heterogeneity. Accurate subtyping requires the simultaneous detection of different biomarkers, which is crucial for personalized treatment strategies. However, existing methodologies are hindered by limited versatility and sensing performance. To overcome these hurdles, this study presents a universal 3D-Hybridization Chain Reaction (3D-HCR) system for RNA detection and subtype-specific diagnosis of breast cancer. The system integrated a universal trigger for HCR, thereby circumventing the need for complex sequence design and enabling the analysis of various RNA targets. Leveraging the spatial-confinement effect offered by DNA nanocarriers, this system exhibited superior amplification efficiency, achieving detection limits of 3.83 pM and 4.96 pM for PD-L1 mRNA and miR-21, respectively. Importantly, the system could differentiate between triple-negative breast cancer and estrogen receptor-positive breast cancer in both living cells and clinical tissues. These findings underscore the potential of the universal 3D-HCR system as a promising tool in clinical diagnostics. With its proven proficiency in breast cancer diagnostics and versatility in RNA analysis, this system holds the promise of broadening the horizons of precision medicine.

What can we learn when fitting a simple telegraph model to a complex gene expression model?

In experiments, the distributions of mRNA or protein numbers in single cells are often fitted to the random telegraph model which includes synthesis and decay of mRNA or protein, and switching of the gene between active and inactive states. While commonly used, this model does not describe how fluctuations are influenced by crucial biological mechanisms such as feedback regulation, non-exponential gene inactivation durations, and multiple gene activation pathways. Here we investigate the dynamical properties of four relatively complex gene expression models by fitting their steady-state mRNA or protein number distributions to the simple telegraph model. We show that despite the underlying complex biological mechanisms, the telegraph model with three effective parameters can accurately capture the steady-state gene product distributions, as well as the conditional distributions in the active gene state, of the complex models. Some effective parameters are reliable and can reflect realistic dynamic behaviors of the complex models, while others may deviate significantly from their real values in the complex models. The effective parameters can also be applied to characterize the capability for a complex model to exhibit multimodality. Using additional information such as single-cell data at multiple time points, we provide an effective method of distinguishing the complex models from the telegraph model. Furthermore, using measurements under varying experimental conditions, we show that fitting the mRNA or protein number distributions to the telegraph model may even reveal the underlying gene regulation mechanisms of the complex models. The effectiveness of these methods is confirmed by analysis of single-cell data for E. coli and mammalian cells. All these results are robust with respect to cooperative transcriptional regulation and extrinsic noise. In particular, we find that faster relaxation speed to the steady state results in more precise parameter inference under large extrinsic noise.

Prediction of response to neoadjuvant chemotherapy by MammaTyper® across breast cancer subtypes: A retrospective cross-sectional study.

BACKGROUND: Neoadjuvant chemotherapy (NACT) is widely used in the treatment of triple-negative and HER2-positive breast cancer (BC), but its use in estrogen receptor (ER) and/or progesterone receptor (PR) positive/HER2-negative BC is questioned because of the low pathologic complete response (pCR) rates. This retrospective study assessed the mRNA-based MammaTyper® assay's capability of predicting pCR with NACT, and ER, PR, Ki67, and HER2 status at immunohistochemical (IHC) through transcriptomics. METHODS: Diagnostic biopsies from 76 BC patients treated at the Cremona Hospital between 2012-2018 were analyzed. Relative mRNA expression levels of ERBB2, ESR1, PGR, and MKI67 were measured using the MammaTyper® kit and integrated into a pCR score. Predicting capability of pCR and standard IHC biomarkers could be assessed with ROC curves in 75 and 76 patients, respectively. RESULTS: Overall, 68.0% patients obtained a MammaTyper® high-score and 32.0% a MammaTyper® low-score. Among high-score patients, 62.7% achieved pCR, compared to 16.7% in the low-score group (p = 0.0003). The binary MammaTyper® score showed good prediction of pCR in the overall cohort (area under curve [AUC] = 0.756) and in HR+/HER2-negative cases (AUC = 0.774). In cases with residual disease, the continuous MammaTyper® score correlated moderately with residual tumor size and decrease in tumor size. MammaTyper® showed substantial agreement with IHC for ESR1/ER and ERBB2/HER2, and moderate agreement for PGR/PR and MKI67/Ki67. CONCLUSION: Overall, MammaTyper® pCR score may serve as a standardized tool for predicting NACT response in HR+/HER2-negative BC, potentially guiding treatment strategies. Additionally, it could provide a more standardized and reproducible assessment of ER, PR, HER2, and Ki67 status.

mRNA Display Pipeline for Protein Biosensor Construction.

Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.

Photoactivated Controlled Dnazyme Platform for on-Demand Activation Sensitive Electrochemiluminescence mRNA Analysis.

Programming ultrasensitive and stimuli-responsive DNAzyme-based probes holds great potential for on-demand biomarker detection. Here, an optically triggered DNAzyme platform was reported for on-demand activation-sensitive electrochemiluminescence (ECL) c-myc mRNA analysis. In this design, the sensing and recognition function of the split DNAzyme (SDz) probe was silent by engineering a blocking sequence containing a photocleavable linker (PC-linker) group at a defined site that could be indirectly cleaved by 302 nm ultraviolet (UV) light. When the SDz probes were assembled on the Au nanoparticles and potassium (K) element doped graphitic carbon nitride nanosheet (K-doped g-C3N4) covered electrode, UV light activation induces the configurational switching and consequently the formation of an active DNAzyme probe with the help of target c-myc mRNA, allowing the cleavage of the substrate strand by magnesium ions (Mg2+). Thus, the release of a ferrocene (Fc)-labeled DNAzyme 2 strand contributed to an extreme ECL signal recovery. In the meantime, the released target c-myc mRNA combined another inactive SDz motif to form active DNAzyme and repeat the cyclic cleavage reaction, resulting in the signal amplification. Furthermore, according to the responses toward two other designed nPC-SDz and m-SDz probes, we demonstrated that controlled UV light mediated photoactivation of the DNAzyme biosensor "on demand" effectively constrained the ECL signal to the mRNA of interest. Moreover, false positive signals could also be avoided due to such a photoactivation design with UV light. Therefore, this study provided a simple methodology that may be broadly applicable for investigating the mRNA-associated physiological events that were difficult to access using traditional DNAzyme probes.

Comprehensive assessment of mRNA isoform detection methods for long-read sequencing data.

The advancement of Long-Read Sequencing (LRS) techniques has significantly increased the length of sequencing to several kilobases, thereby facilitating the identification of alternative splicing events and isoform expressions. Recently, numerous computational tools for isoform detection using long-read sequencing data have been developed. Nevertheless, there remains a deficiency in comparative studies that systemically evaluate the performance of these tools, which are implemented with different algorithms, under various simulations that encompass potential influencing factors. In this study, we conducted a benchmark analysis of thirteen methods implemented in nine tools capable of identifying isoform structures from long-read RNA-seq data. We evaluated their performances using simulated data, which represented diverse sequencing platforms generated by an in-house simulator, RNA sequins (sequencing spike-ins) data, as well as experimental data. Our findings demonstrate IsoQuant as a highly effective tool for isoform detection with LRS, with Bambu and StringTie2 also exhibiting strong performance. These results offer valuable guidance for future research on alternative splicing analysis and the ongoing improvement of tools for isoform detection using LRS data.

The Influence of Tacrolimus on Cellular Morphology, Cellular Viability, Osteogenic Differentiation, and mRNA Expression within Stem Cell Spheroids.

Background and Objectives: Tacrolimus is a macrolide lactone compound derived from the bacterium Streptomyces tsukubensis, widely known as an immunosuppressant. In basic research, the effects of tacrolimus on osteogenic differentiation have been tested using mesenchymal stem cells. In this study, tacrolimus's effects on the cellular survival and osteogenic differentiation of stem cell spheroids were investigated. Materials and Methods: Concave microwells were used to form stem cell spheroids in the presence of tacrolimus at final concentrations of 0 µg/mL, 0.1 µg/mL, 1 µg/mL, 10 µg/mL, and 100 µg/mL. A microscope was used to test cellular vitality qualitatively, and an assay kit based on water-soluble tetrazolium salt was used to measure cellular viability quantitatively. Alkaline phosphatase activity and an anthraquinone dye test for measuring calcium deposits were used to assess osteogenic differentiation. To assess the expression of osteogenic differentiation, a quantitative polymerase chain reaction, Western blot, and RNA sequencing were performed. Results: Spheroids across all concentrations maintained a relatively uniform and spherical shape. Cell viability assay indicated that tacrolimus, up to a concentration of 100 µg/mL, did not significantly impair cell viability within spheroids cultured in osteogenic media. The increase in calcium deposition, particularly at lower concentrations of tacrolimus, points toward an enhancement in osteogenic differentiation. There was an increase in COL1A1 expression across all tacrolimus concentrations, as evidenced by the elevated mean and median values, which may indicate enhanced osteogenic activity. Conclusions: This study showed that tacrolimus does not significantly impact the viability of stem cell spheroids in osteogenic media, even at high concentrations. It also suggests that tacrolimus may enhance osteogenic differentiation, as indicated by increased calcium deposition and COL1A1 expression. These findings advance our understanding of tacrolimus's potential roles in tissue repair, regeneration, and stem cell-based therapeutic applications.

Online Nucleotide Mapping of mRNAs.

Messenger RNA (mRNA) can be sequenced via indirect approaches such as Sanger sequencing and next generation sequencing (NGS), or direct approaches like bottom-up mass spectrometry (MS). Direct sequencing allows the confirmation of RNA modifications. However, the conventional bottom-up MS approach involves time-consuming in-solution digestions that require a large amount of sample, and can lead to the RNase contamination of the LC-MS system and column. Here, we describe a platform that enables online nucleotide mapping of mRNAs via the use of immobilized RNase cartridges and 2D-LC-MS instrumentation. The online approach was compared to conventional offline digestion protocols adapted from two published studies. For this purpose, five model mRNAs of varying lengths (996-4521 nucleotides) and chemistries (unmodified uridine vs 5-methoxyuridine (5moU)) were analyzed. The profiles and sequence coverages obtained after RNase T1 digestion were discussed. The online nucleotide mapping achieved comparable or slightly greater sequence coverage for the 5 mRNAs (5.8-51.5%) in comparison to offline approaches (3.7-50.4%). The sequence coverage was increased to 65.6-85.6 and 69.7-85.0% when accounting for the presence of nonunique digestion products generated by the RNase T1 and A, respectively. The online nucleotide mapping significantly reduced the digestion time (from 15 to <5 min), increased the signal intensity by more than 10-fold in comparison to offline approaches.

Evaluation of Smooth Muscle Myosin Heavy Chain Isoform Expressions in a Buried Penis.

BACKGROUND: A buried penis (BP) is rare in which the penile body is retracted into the prepubic adipose tissue. This research focuses on differences in smooth muscle myosin heavy chain (SMMHC) isoform expressions in the dartos fascia. METHODS: A total of 82 children, 41 of whom had BPs, who applied for circumcision between May and November 2021, were included in the study. The cases were divided into four groups aged ≥6 years (NP6, n = 18) and aged ≤3 years (NP3, n = 17) with normal penile appearance, aged ≥6 years (BP6, n = 23) and aged ≤3 years (BP,n = 24) with a BP. SMMHC isoforms mRNA gene expression analyses were performed by quantitative PCR technique in dartos fascia obtained from foreskin removed by circumcision. RESULTS: Compared to the NP3 group, the SM1 mRNA expressed in the BP6 group was statistically significantly higher (p < 0.005). SM2 mRNA levels expressed in dartos fascia were considerably higher in NP6 and NP3 groups compared to BP6 and BP3 groups (p < 0.001). The SM2/SM1 ratio was 0.85 in the BP6 group and 1.46 in the NP6 group, which was statistically significant (p = 0.006) and increased from 0.87 in the BP3 group to 2.21 in the NP3 group (p < 0.001). CONCLUSION: In a buried penis, there is a difference in the expression of SMMHC isoforms. SM1 is highly expressed, while SM2 decreases, increasing the SM2/SM1 ratio. This causes increased contractility in the smooth muscle, leading to retraction of the penile body. The dartos fascia surrounding it resembles aberrant muscle tissue in boys with a BP. LEVEL OF EVIDENCE: Level III. TYPE OF STUDY: Case-control study.

RNA Analysis in Forensic Molecular Biology.

BACKGROUND: Different types of RNA take on multiple crucial functions in living cells and tissues. Messenger RNA (mRNA) is a temporary molecular carrier of genetic information. Analysis of the composition of all mRNA contained in a cell at a given moment, the so-called transcriptome, enables the determination of the type of cell and its condition, e.g., in pathologically altered states. METHODS: This review is based on pertinent publications retrieved by a selective literature search. RESULTS: The analysis of differential gene expression has already been used in forensic molecular biology to determine the type of tissue contained in biological specimens. It is also being used in criminal investigations to determine the composition of mixed traces of various bodily fluids and/or organ tissues. The method is limited by degradation of the mRNA molecules through environmental influences. The use of newly developed molecular biological methods such as massive parallel sequencing can expand the information obtainable by this investigative method. Current research also addresses the forensic potential of deriving relevant information about the crime-e.g., its timing, or the condition of the involved persons-from the totality of mRNA species present in the specimens. CONCLUSION: Forensic RNA analysis can yield a great deal of relevant information. It is likely to be applicable in a much wider variety of forensic situations in the near future.

[Repair effect of different doses of human umbilical cord mesenchymal stem cells on white matter injury in neonatal rats]. / 不同剂量人脐带间å è´¨å¹²ç»†èƒžç§»æ¤å¯¹æ–°ç”Ÿå¤§é¼ è„‘ç™½è´¨æŸä¼¤çš„ä¿®å¤ä½œç”¨.

OBJECTIVES: To compare the repair effects of different doses of human umbilical cord mesenchymal stem cells (hUC-MSCs) on white matter injury (WMI) in neonatal rats. METHODS: Two-day-old Sprague-Dawley neonatal rats were randomly divided into five groups: sham operation group, WMI group, and hUC-MSCs groups (low dose, medium dose, and high dose), with 24 rats in each group. Twenty-four hours after successful establishment of the neonatal rat white matter injury model, the WMI group was injected with sterile PBS via the lateral ventricle, while the hUC-MSCs groups received injections of hUC-MSCs at different doses. At 14 and 21 days post-modeling, hematoxylin and eosin staining was used to observe pathological changes in the tissues around the lateral ventricles. Real-time quantitative polymerase chain reaction was used to detect the quantitative expression of myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) mRNA in the brain tissue. Immunohistochemistry was employed to observe the expression levels of GFAP and neuron-specific nuclear protein (NeuN) in the tissues around the lateral ventricles. TUNEL staining was used to observe cell apoptosis in the tissues around the lateral ventricles. At 21 days post-modeling, the Morris water maze test was used to observe the spatial learning and memory capabilities of the neonatal rats. RESULTS: At 14 and 21 days post-modeling, numerous cells with nuclear shrinkage and rupture, as well as disordered arrangement of nerve fibers, were observed in the tissues around the lateral ventricles of the WMI group and the low dose group. Compared with the WMI group, the medium and high dose groups showed alleviated pathological changes; the arrangement of nerve fibers in the medium dose group was relatively more orderly compared with the high dose group. Compared with the WMI group, there was no significant difference in the expression levels of MBP and GFAP mRNA in the low dose group (P>0.05), while the expression levels of MBP mRNA increased and GFAP mRNA decreased in the medium and high dose groups. The expression level of MBP mRNA in the medium dose group was higher than that in the high dose group, and the expression level of GFAP mRNA in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the protein expression of GFAP and NeuN in the low dose group (P>0.05), while the expression of NeuN protein increased and GFAP protein decreased in the medium and high dose groups. The expression of NeuN protein in the medium dose group was higher than that in the high dose group, and the expression of GFAP protein in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the number of apoptotic cells in the low dose group (P>0.05), while the number of apoptotic cells in the medium and high dose groups was less than that in the WMI group, and the number of apoptotic cells in the medium dose group was less than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the escape latency time in the low dose group (P>0.05); starting from the third day of the latency period, the escape latency time in the medium dose group was less than that in the WMI group (P<0.05). The medium and high dose groups crossed the platform more times than the WMI group (P<0.05). CONCLUSIONS: Low dose hUC-MSCs may yield unsatisfactory repair effects on WMI in neonatal rats, while medium and high doses of hUC-MSCs have significant repair effects, with the medium dose demonstrating superior efficacy.

Developing an interpretation model for body fluid identification.

Criminal investigations, particularly sexual assaults, frequently require the identification of body fluid type in addition to body fluid donor to provide context. In most cases this can be achieved by conventional methods, however, in certain scenarios, alternative molecular methods are required. An example of this is the detection of menstrual fluid and vaginal material, which are not able to be identified using conventional techniques. Endpoint reverse-transcription PCR (RT-PCR) is currently used for this purpose to amplify body fluid specific messenger RNA (mRNA) transcripts in forensic casework. Real-time quantitative reverse-transcription PCR (RT-qPCR) is a similar method but utilises fluorescent markers to generate quantitative results in the form of threshold cycle (Cq) values. Despite the uncertainty surrounding body fluid identification, most interpretation guidelines utilise categorical statements. Probabilistic modelling is more realistic as it reflects biological variation as well as the known performance of the method. This research describes the application of various machine learning models to single-source mRNA profiles obtained by RT-qPCR and assesses their performance. Multinomial logistic regression (MLR), Naïve Bayes (NB), and linear discriminant analysis (LDA) were used to discriminate between the following body fluid categories: saliva, circulatory blood, menstrual fluid, vaginal material, and semen. We identified that the performance of MLR was somewhat improved when the quantitative dataset of the original Cq values was used (overall accuracy of approximately 0.95) rather than presence/absence coded data (overall accuracy of approximately 0.94). This indicates that the quantitative information obtained by RT-qPCR amplification is useful in assigning body fluid class. Of the three classification methods, MLR performed the best. When we utilised receiver operating characteristic curves to observe performance by body fluid class, it was clear that all methods found difficulty in classifying menstrual blood samples. Future work will involve the modelling of body fluid mixtures, which are common in samples analysed as part of sexual assault investigations.

Development of a capillary zone electrophoresis method to monitor magnesium ion consumption during in vitro transcription for mRNA production.

Messenger RNA (mRNA) vaccines represent a landmark in vaccinology, especially with their success in COVID-19 vaccines, which have shown great promise for future vaccine development and disease prevention. As a platform technology, synthetic mRNA can be produced with high fidelity using in vitro transcription (IVT). Magnesium plays a vital role in the IVT process, facilitating the phosphodiester bond formation between adjacent nucleotides and ensuring accurate transcription to produce high-quality mRNA. The development of the IVT process has prompted key inquiries about in-process characterization of magnesium ion (Mg++) consumption, relating to the RNA polymerase (RNAP) activation, fed-batch mode production yield, and mRNA quality. Hence, it becomes crucial to monitor the free Mg++ concentration throughout the IVT process. However, no free Mg++ analysis method has been reported for complex IVT reactions. Here we report a robust capillary zone electrophoresis (CZE) method with indirect UV detection. The assay allows accurate quantitation of free Mg++ for the complex IVT reaction where it is essential to preserve IVT samples in their native-like state during analysis to avoid dissociation of bound Mg complexes. By applying this CZE method, the relationships between free Mg++ concentration, the mRNA yield, and dsRNA impurity level were investigated. Such mechanistic understanding facilitates informed decisions regarding the quantity and timing of feeding starting materials to increase the yield. Furthermore, this approach can serve as a platform method for analyzing the free Mg++ in complex sample matrices where preserving the native-like state of Mg++ binding is key for accurate quantitation.