In Vitro Embryogenesis and Gastrulation Using Stem Cells in Mice and Humans.

During early mammalian embryonic development, fertilized one-cell embryos develop into pre-implantation blastocysts and subsequently establish three germ layers through gastrulation during post-implantation development. In recent years, stem cells have emerged as a powerful tool to study embryogenesis and gastrulation without the need for eggs, allowing for the generation of embryo-like structures known as synthetic embryos or embryoids. These in vitro models closely resemble early embryos in terms of morphology and gene expression and provide a faithful recapitulation of early pre- and post-implantation embryonic development. Synthetic embryos can be generated through a combinatorial culture of three blastocyst-derived stem cell types, such as embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm cells, or totipotent-like stem cells alone. This review provides an overview of the progress and various approaches in studying in vitro embryogenesis and gastrulation in mice and humans using stem cells. Furthermore, recent findings and breakthroughs in synthetic embryos and gastruloids are outlined. Despite ethical considerations, synthetic embryo models hold promise for understanding mammalian (including humans) embryonic development and have potential implications for regenerative medicine and developmental research.

Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance.

The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.

CRISPR/Cas9-Mediated Customizing Strategies for Adoptive T-Cell Therapy.

Clustered regularly interspaced short palindromic repeat-associated protein Cas9 (CRISPR/Cas9) technology is at the forefront of cancer immunotherapy innovation, offering precise and personalized treatment strategies. In this review, we discuss CRISPR/Cas9's ability to precisely edit the genome, its impact on immune checkpoint control, and its application in immune cell engineering, where it surpasses traditional gene editing techniques. Originally inspired by bacterial defense mechanisms, this technology has made great strides in cancer immunotherapy as a mechanism to specifically target the PD-1/PD-L1 pathway in immune checkpoint blockades. In addition, CRISPR/Cas9 plays an important role in cancer treatment by facilitating genetic modifications to enhance the properties of adoptive cell therapy, optimizing the therapeutic potential of this approach. This review provides an overview of the development of CRISPR/Cas9, its important role in immune checkpoint control, applications in immune cell engineering, and the current status of clinical trials. However, safety concerns related to off-target effects and unintended mutations require continued research and caution. Continued advances in CRISPR technology hold the promise of revolutionizing the cancer treatment paradigm, providing personalized and effective therapies for patients with various types of cancer.

An Antibody-CRISPR/Cas Conjugate Platform for Target-Specific Delivery and Gene Editing in Cancer.

The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody-CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2-positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2. The resultant antibody-conjugated CRISPR/Cas nanocomplexes can be specifically delivered and induce gene editing in HER2-positive cancer cells in vitro. It is demonstrated that the in vivo delivery of the antibody-CRISPR/Cas nanocomplexes can effectively disrupt the plk1 gene in HER2-positive ovarian cancer, resulting in substantial suppression of tumor growth. The current study presents a useful therapeutic platform for antibody-mediated delivery of CRISPR/Cas for the treatment of various cancers and genetic diseases.

Caspase Cleavage of Receptor Tyrosine Kinases in the Dependence Receptor Family.

Dependence receptors are a group of receptor proteins with shared characteristics of transducing two different signals within cells. They can transduce a positive signal of survival and differentiation in the presence of ligands. On the other hand, dependence receptors can transduce an apoptosis signal in the absence of ligands. The function of these receptors depends on the availability of their ligands. Several receptor tyrosine kinases (RTKs) have been reported as dependence receptors. When cells undergo apoptosis by dependence receptors, the intracellular domain of some RTKs is cleaved by the caspases. Among the RTKs that belong to dependence receptors, we focused on eight RTKs (RET, HER2, MET, ALK, TrkC, EphA4, EphB3, and c-KIT) that are cleaved by caspases. In this review, we describe the features of the receptors, their cleavage sites, and the fate of the cleaved products, as well as recent implications on them being used as potential therapeutics for cancer treatment.

Simple and Multiplexed Detection of Nucleic Acid Targets Based on Fluorescent Ring Patterns and Deep Learning Analysis.

Simple diagnostic tests for nucleic acid targets can provide great advantages for applications such as rapid pathogen detection. Here, we developed a membrane assay for multiplexed detection of nucleic acid targets based on the visualization of two-dimensional fluorescent ring patterns. A droplet of the assay solution is applied to a cellulose nitrate membrane, and upon radial chromatographic flow and evaporation of the solvent, fluorescent patterns appear under UV irradiation. The target nucleic acid is isothermally amplified and is immediately hybridized with fluorescent oligonucleotide probes in a one-pot reaction. We established the fluorescent ring assay integrated with isothermal amplification (iFluor-RFA = isothermal fluorescent ring-based radial flow assay), and feasibility was tested using nucleic acid targets of the receptor binding domain (RBD) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2. We demonstrate that the iFluor-RFA method is capable of specific and sensitive detection in the subpicomole range, as well as multiplexed detection even in complex solutions. Furthermore, we applied deep learning analysis of the fluorescence images, showing that patterns could be classified as positive or negative and that quantitative amounts of the target could be predicted. The current technique, which is a membrane pattern-based nucleic acid assay combined with deep learning analysis, provides a novel approach in diagnostic platform development that can be versatilely applied for the rapid detection of infectious pathogens.

A Lateral Flow Assay for Nucleic Acid Detection Based on Rolling Circle Amplification Using Capture Ligand-Modified Oligonucleotides.

We introduce a lateral flow assay (LFA) integrated with a modified isothermal nucleic acid amplification procedure for rapid and simple genetic testing. Padlock probes specific for the target DNA were designed for ligation, followed by rolling circle amplification (RCA) using capture ligand-modified oligonucleotides as primers. After hybridization with detection linker probes, the amplified target DNA is flowed through an LFA membrane strip for binding of gold nanoparticles as the substrate for colorimetric detection. We established and validated the "RCA-LFA" method for detection of mecA, the antibiotic resistance gene for methicillin-resistant Staphylococcus aureus (MRSA). The assay was optimized using various concentrations of primers and probes for RCA and LFA, respectively. The sensitivity was determined by performing RCA-LFA using various amounts of mecA target DNA, showing a detection limit of ~ 1.3 fmol. The specificity of the assay was examined using target DNAs for other resistance genes as the controls, which demonstrated positive detection signals only for mecA DNA, when added either individually or in combinations with the control targets. Furthermore, applying the RCA-LFA method using specifically designed probes for RNA-dependent RNA polymerase (RdRp) and receptor binding domain (RBD) gene for SARS-CoV-2, which demonstrated feasibility of the method for viral gene targets. The current method suggests a useful platform which can be universally applied for various nucleic acid targets, allowing rapid and sensitive diagnosis at point-of-care. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00080-1.

Simple visualized readout of suppressed coffee ring patterns for rapid and isothermal genetic testing of antibacterial resistance.

We present a facile method based on the coffee ring effect that can rapidly detect antibiotic-resistant bacteria, as an affordable genetic testing platform. When a colloidal solution of particles is dropped onto a substrate surface, an outward capillary flow upon evaporation induces the migration of the particles to the periphery of the droplet, forming a characteristic ring pattern. Herein, we utilize capture DNA microbeads which in the presence of target nucleic acid, form suppressed ring patterns by hybridization-induced crosslinking of the microbeads. The coffee ring-based assay is integrated with isothermal amplification based on rolling circle amplification (RCA), to produce long, single-stranded target DNA and induce hybridization, via a one-step procedure (i-CoRi assay). The resultant ring patterns can be simply observed with the naked eye or recorded with a standard mobile device for readout. The i-CoRi assay was validated for the rapid and specific detection of the antibiotic resistance gene mecA for MRSA, showing that detection was possible at the sub-zeptomolar range (~0.2 zM) with the specificity of distinguishing 2 mismatched bases. The spatial patterns of the microbeads were characterized, showing the dense packing of the microbeads at the center of the droplet and thinning of the ring pattern for the MRSA target, which were distinct from the negative controls MSSA, E. coli, and P. aeruginosa. The images of the microbead patterns were also processed by a simple readout algorithm to discriminate the presence or absence of the coffee ring, to enable diagnostic decision making. The current method provides a rapid and versatile platform for the specific identification of bacterial pathogens and multidrug resistance, especially for diagnosis in resource-limited settings.

Ultra-fast and universal detection of Gram-negative bacteria in complex samples based on colistin derivatives.

Gram-negative bacteria are a significant cause of infections acquired in both hospital and community settings, resulting in a high mortality rate worldwide. Currently, a Gram-negative infection is diagnosed by symptom evaluation and is treated with empiric antibiotics which target both Gram-negative and Gram-positive bacteria. A rapid and simple diagnostic method would enable immediate and targeted treatment, while dramatically reducing antibiotic overuse. Herein, we introduce a method utilizing a fluorescent derivative of colistin (COL-FL), that can directly label the Gram-negative cell wall of live bacteria and universally detect the targets within 10 min. By using the COL-FL assay, we achieved the differential labeling of various Gram-negative pathogens related to hospital-acquired infections, which could be subsequently detected via spectrofluorometry and microscopy. Further, we determined that our method can be used for complex samples, such as combinations of multiple bacterial types; bacteria in the presence of mammalian cells; and bacteria with serum components. This assay can be integrated into a simple diagnostic platform for rapid screening tests and the stratification of Gram-negative bacterial infections in the clinic.

Acute cholecystitis in elderly patients after hip fracture: Incidence and epidemiology.

AIM: Acute cholecystitis is a medical complication that can develop in the postoperative period after hip surgery. However, few studies have examined this complication in elderly patients. Our aim was therefore to evaluate the incidence and clinical manifestations of acute cholecystitis after hip fracture in elderly patients. METHODS: Medical records and radiological studies of patients aged older than 65 years who underwent hip surgery for femoral neck or intertrochanteric fractures at a single hospital from April 2003 to March 2013 were reviewed retrospectively. We analyzed the type of cholecystitis (acalculous or calculous), clinical manifestations, fracture type (neck or trochanteric fracture), age, sex, body mass index, type of surgery, time to surgery, time from surgery to onset of acute cholecystitis and the timing of ambulation in acute cholecystitis cases. RESULTS: There were nine confirmed acute cholecystitis cases among 1211 hip fractures; thus, the incidence of acute cholecystitis within 2 months after hip fracture surgery was 0.74%. CONCLUSIONS: The incidence of acute cholecystitis was higher than we expected, and this condition can lead to more serious problems if overlooked. Acute cholecystitis as a medical complication after hip fracture was underestimated in previous studies. Furthermore, acute cholecystitis should be considered as a complication of hip fracture, not hip surgery, in the elderly. The present study does not imply that hip fracture causes acute cholecystitis, although elderly hip fracture patients are in an extremely debilitated state and are prone to developing acute cholecystitis.

Red Ginseng-containing diet helps to protect mice and ferrets from the lethal infection by highly pathogenic H5N1 influenza virus.

The highly pathogenic (HP) H5N1 influenza virus is endemic in many countries and has a great potential for a pandemic in humans. The immune-enhancing prowess of ginseng has been known for millennia. We aimed to study whether mice and ferrets fed with Red Ginseng could be better protected from the lethal infections of HP H5N1 influenza virus than the infected unfed mice and ferrets. We fed mice and ferrets with Red Ginseng prior to when they were infected with HP H5N1 influenza virus. The mice and ferrets fed with a 60-day diet containing Red Ginseng could be protected from lethal infections by HP H5N1 influenza virus (survival rate of up to 45% and 40%, respectively). Interferon-α and -γ antiviral cytokines were significantly induced in the lungs of mice fed Red Ginseng, compared to mice fed an unsupplemented diet. These data suggest that the diet with the immune-enhancing Red Ginseng could help humans to overcome the infections by HP H5N1 influenza virus.

Severe pathogenesis of influenza B virus in pregnant mice.

The study on pathogenesis of influenza B virus during pregnancy is limited. Here, we showed using a mouse model that influenza B virus could cause severe disease including death during pregnancy. Infected pregnant mice resulted in 40% mortality, but infected age-matched non-pregnant mice did not show any death. Infected pregnant mice contained high viral loads in lungs with the elevated inductions of inflammatory cytokines and chemokines than infected non-pregnant mice. Infected pregnant mice delivered lower number of neonates than uninfected pregnant mice, suggesting adverse effects of influenza B virus on fetuses. Progesterone which is important for maintaining pregnancy was reduced in uteruses of infected pregnant mice than in those of uninfected pregnant mice. Taken together, our results suggest that influenza B virus can cause severe disease during pregnancy, and that preventive measures including vaccination may be important for protecting women during pregnancy.