Identification and biochemical characterization of a novel halolysin from Halorubellus sp. PRR65 with a relatively high temperature activity.

Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp154-His193-Ser348. Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The Km, Vmax and Kcat for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg-1 and 1538.09 S-1, respectively, under 60 °C, pH 8.0 and 4.0 M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities.

Modified Ce/Zr-MOF Nanoparticles Loaded with Curcumin for Alzheimer's Disease via Multifunctional Modulation.

Introduction: Alzheimer's disease (AD), a neurodegenerative condition, stands as the most prevalent form of dementia. Its complex pathological mechanisms and the formidable blood-brain barrier (BBB) pose significant challenges to current treatment approaches. Oxidative stress is recognized as a central factor in AD, underscoring the importance of antioxidative strategies in its treatment. In this study, we developed a novel brain-targeted nanoparticle, Ce/Zr-MOF@Cur-Lf, for AD therapy. Methods: Layer-by-layer self-assembly technology was used to prepare Ce/Zr-MOF@Cur-Lf. In addition, the effect on the intracellular reactive oxygen species level, the uptake effect by PC12 and bEnd.3 cells and the in vitro BBB permeation effect were investigated. Finally, the mouse AD model was established by intrahippocampal injection of Aß1-42, and the in vivo biodistribution, AD therapeutic effect and biosafety of the nanoparticles were researched at the animal level. Results: As anticipated, Ce/Zr-MOF@Cur-Lf demonstrated efficient BBB penetration and uptake by PC12 cells, leading to attenuation of H2O2-induced oxidative damage. Moreover, intravenous administration of Ce/Zr-MOF@Cur-Lf resulted in rapid brain access and improvement of various pathological features of AD, including neuronal damage, amyloid-ß deposition, dysregulated central cholinergic system, oxidative stress, and neuroinflammation. Conclusion: Overall, Ce/Zr-MOF@Cur-Lf represents a promising approach for precise brain targeting and multi-target mechanisms in AD therapy, potentially serving as a viable option for future clinical treatment.

Efficient, High-Quality Engineering of Therapeutic Extracellular Vesicles on an Integrated Nanoplatform.

Engineered extracellular vesicles (EVs) have been recognized as important therapeutics for gene and cell therapy. To achieve clinically desired therapy, technologies for EV engineering have high demands on the efficacy in producing EVs and their qualities, which, however, remain challenging to conventional routes due to their limited control on therapeutic payload delivery, EV secretion, and extracellular microenvironments. Here, we report a nanoplatform (denoted as PURE) that enables efficient electro-transfection while stimulating cells to produce high-quality EVs carrying functional RNAs. PURE further employs an ammonium removal zone to maintain the physiological conditions of the extracellular microenvironment and an EV uptake zone that efficiently (87.1%) captures EVs in situ with porous hydrogels. The platform achieved about a 12-fold higher yield of engineered EVs and a 146-fold abundance of desired therapeutics compared to those naturally secreted from cells. The PURE-engineered miR-130a-EVs were validated for effectively upregulating the mTOR signaling pathway in both in vitro and in vivo. Their therapeutic capability was then verified by enhancing the in vitro activation of primordial follicles. In vivo applications further highlighted the therapeutic effects of miR-130a-EVs in restoring ovary function in aged mice. The PURE platform represents a strategy for the clinical translation of EV-mediated therapy.

A Degradable Bioelectronic Scaffold for Localized Cell Transfection toward Enhancing Wound Healing in a 3D Space.

Large skin wounds, with extensive surface area and deep vertical full-thickness involvement, can pose significant challenges in clinical settings. Traditional routes for repairing skin wounds encompass three hallmarks: 1) scab formation for hemostasis; 2) proliferation and migration of epidermal cells for wound closure; 3) proliferation, migration, and functionalization of fibroblasts and endothelial cells for dermal remodeling. However, this route face remarkable challenges to healing large wounds, usually leading to disordered structures and loss of functions in the regenerated skin, due to limited control on the transition among the three stages. In this work, an implantable bioelectronics is developed that enables the synchronization of the three stages, offering accelerated and high-quality healing of large skin wounds. The system efficiently electro-transfect local cells near the wounds, forcing cellular proliferation, while providing a 3D porous environments for synchronized migration of epidermal and dermal cells. In vivo experiments demonstrated that the system achieved synchronous progression of multiple layers within the wounds, leading to the reconstruction of a complete skin structure similar to healthy skin, which presents a new avenue for the clinical translation of large wound healing.

Structural and functional insights into the helicase protein E5 of Mpox virus.

Mpox virus (MPXV) can cause mpox in humans. Due to its quick and wide spread in the past two years, mpox has turned into a significant public health concern. Helicase E5 is a multi-domain protein; its primer synthesis and DNA unwinding activity are required for genome uncoating and DNA replication of MPXV. However, the in vitro DNA unwinding activity has never been demonstrated. Here, we report the structural and biochemical studies of MPXV E5, showing that the full-length protein adopts an auto-inhibited conformation. Truncation of the N-terminus can recover the in vitro unwinding activity of E5 towards the forked DNA. Further structural analysis reveals that MPXV E5 shares a conserved mechanism in DNA unwinding and primer synthesis with the homologous proteins. These findings not only advance our understanding on the function of MPXV E5, but also provide a solid basis for the development of anti-poxvirus drugs.

Advanced micro/nano-electroporation for gene therapy: recent advances and future outlook.

Gene therapy is a promising disease treatment approach by editing target genes, and thus plays a fundamental role in precision medicine. To ensure gene therapy efficacy, the effective delivery of therapeutic genes into specific cells is a key challenge. Electroporation utilizes short electric pulses to physically break the cell membrane barrier, allowing gene transfer into the cells. It dodges the off-target risks associated with viral vectors, and also stands out from other physical-based gene delivery methods with its high-throughput and cargo-accelerating features. In recent years, with the help of advanced micro/nanotechnology, micro/nanostructure-integrated electroporation (micro/nano-electroporation) techniques and devices have significantly improved cell viability, transfection efficiency and dose controllability of the electroporation strategy, enhancing its application practicality especially in vivo. This technical advancement makes micro/nano-electroporation an effective and versatile tool for gene therapy. In this review, we first introduce the evolution of electroporation technique with a brief explanation of the perforation mechanism, and then provide an overview of the recent advancements and prospects of micro/nano-electroporation technology in the field of gene therapy. To comprehensively showcase the latest developments of micro/nano-electroporation technology in gene therapy, we focus on discussing micro/nano-electroporation devices and current applications at both in vitro and in vivo levels. Additionally, we outline the ongoing clinical studies of gene electrotransfer (GET), revealing the tremendous potential of electroporation-based gene delivery in disease treatment and healthcare. Lastly, the challenges and future directions in this field are discussed.

Upstream nitrogen availability determines the Microcystis salt tolerance and influences microcystins release in brackish water.

The occurrence of large Microcystis biomass in brackish waters is primarily caused by its downward transportation from the upstream freshwater lakes and reservoirs through rivers rather than due to in situ bloom formation. Factors that determine the survival of freshwater cyanobacteria in brackish waters have not been well investigated. Here, we studied the spatiotemporal variability of inorganic nitrogen in an upstream lake and conducted laboratory and in-situ experiments to assess the role of nitrogen availability on the salt tolerance of Microcystis and the release of microcystins. A series of field experiments were carried out during bloom seasons to evaluate the salt tolerance of natural Microcystis colonies. The salt tolerance threshold varied from 7 to 17 and showed a positive relationship with intracellular carbohydrate content and a negative relationship with nitrogen availability in water. In August when upstream nitrogen availability was lower, the Microcystis colonies could maintain their biomass even after a sudden increase in salinity from 4 to 10. Laboratory-cultivated Microcystis that accumulated higher carbohydrate content at lower nitrogen availability showed better cell survival at higher salinity. The sharp release of microcystins into the surrounding water occurred when salinity exceeded the salt tolerance threshold of the Microcystis. Thus, Microcystis with higher salt tolerance can accumulate more toxins in cells. The obtained results suggest that the cell survival and toxin concentration in brackish waters depend on the physiological properties of Microcystis formed in the upstream waters. Thus, the life history of Microcystis in upstream waters could have a significant impact on its salt tolerance in downstream brackish waters, where the ecological risk of the salt-tolerant Microcystis requires special and careful management in summer at low nitrogen availability.

Recent Advances in Bioelectronics for Localized Drug Delivery.

The last decade has witnessed remarkable advancements in bioelectronics, ushering in a new era of wearable and implantable devices for drug delivery. By utilizing miniaturized system design and/or flexible materials, bioelectronics illustrates ideal integration with target organs and tissues, making them ideal platforms for localized drug delivery. Furthermore, the development of electrically assisted drug delivery systems has enhanced the efficiency and safety of therapeutic administration, particularly for the macromolecules that encounter additional challenges in penetrating biological barriers. In this review, a concise overview of recent progress in bioelectronic devices for in vivo localized drug delivery, with highlights on the latest trends in device design, working principles, and their corresponding functionalities, is provided. The reported systems based on their targeted delivery locations as wearable systems, ingestible systems, and implantable systems are categorized. Each category is introduced in detail by highlighting the special requirements for devices and the corresponding solutions. The remaining challenges in this field and future directions are also discussed.

The Trends and Hotspots in Premature Ovarian Insufficiency Therapy from 2000 to 2022.

This study aims to map the knowledge structure and themes trends of primary ovarian insufficiency (POI) therapy to help researchers rapidly master the hotspots and prospects of POI therapy from the increasing number of publications. The literature search and bibliometric analyses were performed by using Web of Science Core Collection and VOSviewer. Annual publications from 2000 to 2022 continued to increase with some fluctuations. The most productive country, organization, and journal were the USA, Shanghai Jiao Tong University, and Human Reproduction, respectively. Harvard University was the organization with the highest citation. Fertility and Sterility and Nelson, L.M. were the most influential journal and author, respectively. Seven clusters separated by keywords association showed the extensive scope of POI therapy. The hotspots of POI therapy were hormone replacement therapy and fertility preservation, and the innovative treatment strategies including in vitro activation and mesenchymal stem cells had development potential. In addition, our result showed that the high-cited articles were published in journals with high impact factors. The paper provides a comprehensive overview of the development and hotspots of POI therapy, allowing researchers to recognize the current status and future directions of POI therapy.

Nanoconfinement effect based in-fiber extraction and derivatization method for ultrafast analysis of twenty amines in human urine by GC-MS: Application to cancer diagnosis biomarkers' screening.

Amines, including amino acids and biogenic amines are widely recognized as cancer biomarkers for early diagnosis and treatment. But rapid analysis of amines in biological samples with high sensitivity, good durability and low cost is still challenging. Herein, we proposed an ultrafast biological sample preparation method based on in-fiber extraction and derivatization using a homemade needle-tip devise. Under the nanoconfinement effect, the extraction was completed within 2 min in nanofibers, followed by the in-suit derivatization within 3 min under room temperature using pentafluoropropionic anhydride (PFA) as derivatization reagent, then the needle was inserted into the inlet of GC-MS directly for thermal desorption to enable the complete injection. Additionally, the established sample preparation strategy consumed only 20 µL organic solvent and urine sample, and the sensitivity was increased compared with other methods. After systematical optimization and validation, coupled to GC-MS, the established method was successfully applied to quantify twenty amines in urine of healthy people and lung, colorectal and breast cancer patients. The diagnosis biomarkers for each cancer were screened out and accessed by multiple statistical approaches. Then we further constructed the cancer diagnosis model of each cancer through binary logistic regression analysis for clinical use. Taken together, the established nanoconfinement effect based in-fiber extraction and derivatization biological sample preparation strategy in combination with GC-MS was an ultrafast method for biological analysis with high sensitivity, good durability, little organic solvent consumption and low cost. It possesses great potential in clinical cancer screening and treatment.

Highly integrated watch for noninvasive continual glucose monitoring.

This article reports a highly integrated watch for noninvasive continual blood glucose monitoring. The watch employs a Nafion-coated flexible electrochemical sensor patch fixed on the watchband to obtain interstitial fluid (ISF) transdermally at the wrist. This reverse iontophoresis-based extraction method eliminates the pain and inconvenience that traditional fingerstick blood tests pose in diabetic patients' lives, making continual blood glucose monitoring practical and easy. All electronic modules, including a rechargeable power source and other modules for signal processing and wireless transmission, are integrated onto a watch face-sized printed circuit board (PCB), enabling comfortable wearing of this continual glucose monitor. Real-time blood glucose levels are displayed on the LED screen of the watch and can also be checked with the smartphone user interface. With 23 volunteers, the watch demonstrated 84.34% clinical accuracy in the Clarke error grid analysis (zones A + B). In the near future, commercial products could be developed based on this lab-made prototype to provide the public with noninvasive continual glucose monitoring.

Research on dual innovation incentive mechanism in terms of organizations' differential knowledge absorptive capacity.

Differences in the capacity for absorption between different organizations will have an important impact on an organization's choices of innovation exploration and exploitive innovation strategies. Organizations need to explore correct strategic decisions under different policies for long-term development. This study with limited rational first-mover and late-mover organizations as the research object, based on the evolutionary game theory model, using visualization system deduced first-mover and late-mover organizations in the knowledge absorptive capacity differences and incentive policies under the condition of different strategies selection process. The research shows that the rationality of policy incentive setting has a direct impact on the choice of organizational dual innovation strategy with different knowledge absorption capacities. The market pattern is stable and organizational knowledge absorption capacity is different. The higher the policy incentive level is, the more the organization is inclined to carry out exploratory innovation activities. Under the environment of stable market structure, different organizational knowledge absorption capacity, and no policy incentive, late-mover cannot adopt exploratory innovation strategy alone. When the market pattern is stable and the absorptive capacity of the organization is different, whether the late-mover can adopt the exploratory innovation strategy depends on the policy incentive level. In this case, the optimal situation is to have the opportunity to change to exploratory innovation at the same time as the first-movers.

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Endogenous Protection from Ischemic Brain Injury by Preconditioned Monocytes.

Exposure to low-dose lipopolysaccharide (LPS) before cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning (PC). Although it is well established that LPS-PC induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by PC and tested whether monocytes may be reprogrammed by ex vivo LPS exposure, thus modulating inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose LPS induces a Ly6Chi monocyte response that protects the brain after transient middle cerebral artery occlusion (MCAO) in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 h after transient MCAO reduced brain injury. Gene expression and functional studies showed that IL-10, inducible nitric oxide synthase, and CCR2 in monocytes are essential for neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to LPS and then injected into male mice after stroke. Cell-tracking studies showed that protective monocytes are mobilized from the spleen and reach the brain and meninges, where they suppress postischemic inflammation and neutrophil influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable of protecting the brain with an extended therapeutic window and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes in patients after ischemic stroke.SIGNIFICANCE STATEMENT Inflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using lipopolysaccharide (LPS) preconditioning (PC) as an approach to induce ischemic tolerance in mice, we found generation of neuroprotective monocytes within the spleen, from which they traffic to the brain and meninges, suppressing postischemic inflammation. Importantly, systemic LPS-PC can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of BM mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.