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Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1–10 μl) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections. [ABSTRACT FROM AUTHOR]

Of all methods exercised in modern molecular biology, modification of cellular properties through the introduction or removal of nucleic acids is one of the most fundamental. As such, several methods have arisen to promote this process; these include the condensation of nucleic acids with calcium, polyethylenimine or modified lipids, electroporation, viral production, biolistics, and microinjection. An ideal transfection method would be (1) low cost, (2) exhibit high levels of biological safety, (3) offer improved efficacy over existing methods, (4) lack requirements for ongoing consumables, (5) work efficiently at any scale, (6) work efficiently on cells that are difficult to transfect by other methods, and (7) be capable of utilizing the widest array of existing genetic resources to facilitate its utility in research, biotechnical and clinical settings. To address such issues, we describe here Pressure-jump-poration (PJP), a method using rapid depressurization to transfect even difficult to modify primary cell types such as embryonic stem cells. The results demonstrate that PJP can be used to introduce an array of genetic modifiers in a safe, sterile manner. Finally, PJP-induced transfection in primary versus transformed cells reveals a surprising dichotomy between these classes which may provide further insight into the process of cellular transformation. [ABSTRACT FROM AUTHOR]

In this study, extended-spectrum β-lactamase (ESBL) Escherichia coli were isolated from five dairy farms in three areas of northern Xinjiang, China. Molecular biological techniques were used to systematically analyze drug resistance phenotypes and genotypes, virulence genes, phylogenetics, biofilm formation (BF), and pulsed field gel electrophoresis (PFGE) typing of isolated ESBL E. coli strains. A total of 766 samples were collected from five dairy farms in Shihezi, Urumqi and Yili, from which 149 (19.5 %, 95 % CI: 16.65 %–22.25 %) ESBL E. coli strains were isolated. Their distribution and contamination levels varied from region to region, with 16.2 % (68/419) in Urumqi, 22.4 % (60/268) in Shihezi, and 26.6 % (21/79) in Yili. The majority of isolates (97.3 %, 145/149) harbored the β-lactamase bla CTX-M gene; while bla CTX-M-1 was the dominant phylogenetic group. The analysis of 21 resistance genes and the susceptibility to 13 different antibiotics showed that 91.3 % (136/149) of strains were resistant to three or more antibiotics. Thirty-six strains (24.2 %) belonged to extraintestinal pathogenic E. coli (ExPEC), and phylogenetic typing results were mainly grouped A (50.3 %) and B1 (37.6 %). Also, the biofilm assay revealed that 112 strains (75.2 %) could form biofilms. PFGE results showed that the 49 isolates revealed 21 major pulsotypes (P1–P21) and 28 subtypes with 80 % similarity, indicating the overall genetic diversity in the distribution area and sources of the samples. • 91.3 % of the E. coli strains were classified as multi-resistant strains, and carried multiple drug resistance genes. • Of the 149 E. coli isolates, 36 were defined as ExPEC, indicating their pathogenic potential. • The coincidence rate in 149 E. coli strains for BF ability and antibiotic resistance (13 antibiotics) was >77.2 %. • PFGE showed 21 major pulsotypes indicating the genetic diversity of isolates in terms of distribution region and source. [ABSTRACT FROM AUTHOR]

Emotion recognition is one of the most important research directions in the field of brain–computer interface (BCI). However, to conduct electroencephalogram (EEG)-based emotion recognition, there exist difficulties regarding EEG signal processing; moreover, the performance of classification models in this regard is restricted. To counter these issues, the 2022 World Robot Contest successfully held an affective BCI competition, thus promoting the innovation of EEG-based emotion recognition. In this paper, we propose the Transformer-based ensemble (TBEM) deep learning model. TBEM comprises two models: a pure convolutional neural network (CNN) model and a cascaded CNN-Transformer hybrid model. The proposed model won the abovementioned affective BCI competition's final championship in the 2022 World Robot Contest, demonstrating the effectiveness of the proposed TBEM deep learning model for EEG-based emotion recognition. [ABSTRACT FROM AUTHOR]

The primary forms of cell death seen in ischemic stroke are of two major types: a necrotic/necroptotic form, and an apoptotic form that is frequently seen in penumbral regions of injury. Typically apoptotic versus necroptotic programmed cell death is described as competitive in nature, where necroptosis is often described as playing a backup role to apoptosis. In the present study, we examined the relationship between these two forms of cell death in a murine endothelin-1 model of ischemia–reperfusion injury in wildtype and caspase-3 null mice with and without addition of the pharmacologic RIPK1 phosphorylation inhibitor necrostatin-1. Analyses of ischemic brain injury were performed via both cellular and volumetric assessments, electron microscopy, TUNEL staining, activated caspase-3 and caspase-7 staining, as well as CD11b and F4/80 staining. Inhibition of caspase-3 or RIPK1 phosphorylation demonstrates significant neural protective effects which are non-additive and exhibit significant overlap in protected regions. Interestingly, morphologic analysis of the cortex demonstrates reduced apoptosis following RIPK1 inhibition. Consistent with this, RIPK1 inhibition reduces the levels of both caspase-3 and caspase-7 activation. Additionally, this protection appears independent of secondary inflammatory mediators. Together, these observations demonstrate that the necroptotic protein RIPK1 modifies caspase-3/-7 activity, ultimately resulting in decreased neuronal apoptosis. These findings thus modify the traditional exclusionary view of apoptotic/necroptotic signaling, revealing a new form of interaction between these dominant forms of cell death. [ABSTRACT FROM AUTHOR]

Cisplatin is a member of a widely utilized class of chemotherapeutic agent that initiates DNA damage response, cell cycle arrest, and p53-dependent apoptotic cell death in concert with DNA‑platinum adduct formation. While normal programmed cell death (PCD) can occur in the developing neuroepithelium in the absence of caspase-3 within certain genetic backgrounds, we observed an absolute dependency upon this executioner caspase with respect to cisplatin-induced PCD in the developing central nervous system (CNS). We therefore examined the nature of this genotoxic injury in the CNS in vivo, in which cisplatin treatment causes widespread cellular injury consistent with hallmarks of apoptosis which are averted upon caspase-3 inhibition. Examination of cisplatin-mediated injury as a function of time revealed the presence of an alternative, delayed form of necroptosis-like cell death which manifests in Casp3 −/− neuroepithelia for several days following the normal pattern of apoptosis. Together, these findings suggest a coordinated regulation of these disparate PCD pathways in response to genotoxic stress in vivo and highlight the unique and critical role which caspase-3 plays among executioner caspases in coordinating apoptotic versus necroptotic responsiveness of the developing CNS to genotoxic injury. • In utero cisplatin treatment causes apoptosis in the developing neuroepithelium. • Casp3 deletion completely prevents this cisplatin-induced apoptotic cell death. • Delayed necroptotic cell death occurs in some cells only in the absence of caspase-3. [ABSTRACT FROM AUTHOR]