PROTAC-mediated vimentin degradation promotes terminal erythroid differentiation of pluripotent stem cells.

BACKGROUND: Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can undergo erythroid differentiation, offering a potentially invaluable resource for generating large quantities of erythroid cells. However, the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors, with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms. METHODS: In this study, we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies, including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs, morphological analysis, quantitative real-time PCR (qRT-PCR), western blotting, flow cytometry, karyotyping, cytospin, Benzidine-Giemsa staining, immunofluorescence assay, and high-speed cell imaging analysis, were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation, as well as their differentiated erythroid cells. RESULTS: Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells, whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin. CONCLUSIONS: VIM disappear during the normal maturation of erythroid cells, whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform, we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization.

Genome-Wide Analysis and Expression Profiling of Soybean RbcS Family in Response to Plant Hormones and Functional Identification of GmRbcS8 in Soybean Mosaic Virus.

Rubisco small subunit (RbcS), a core component with crucial effects on the structure and kinetic properties of the Rubisco enzyme, plays an important role in response to plant growth, development, and various stresses. Although Rbcs genes have been characterized in many plants, their muti-functions in soybeans remain elusive. In this study, a total of 11 GmRbcS genes were identified and subsequently divided into three subgroups based on a phylogenetic relationship. The evolutionary analysis revealed that whole-genome duplication has a profound effect on GmRbcSs. The cis-acting elements responsive to plant hormones, development, and stress-related were widely found in the promoter region. Expression patterns based on the RT-qPCR assay exhibited that GmRbcS genes are expressed in multiple tissues, and notably Glyma.19G046600 (GmRbcS8) exhibited the highest expression level compared to other members, especially in leaves. Moreover, differential expressions of GmRbcS genes were found to be significantly regulated by exogenous plant hormones, demonstrating their potential functions in diverse biology processes. Finally, the function of GmRbcS8 in enhancing soybean resistance to soybean mosaic virus (SMV) was further determined through the virus-induced gene silencing (VIGS) assay. All these findings establish a strong basis for further elucidating the biological functions of RbcS genes in soybeans.

Impact of Different Red Blood Cell Storage Solutions and Conditions on Cell Function and Viability: A Systematic Review.

Red blood cell (RBC) storage solutions have evolved significantly over the past decades to optimize the preservation of cell viability and functionality during hypothermic storage. This comprehensive review provides an in-depth analysis of the effects of various storage solutions and conditions on critical RBC parameters during refrigerated preservation. A wide range of solutions, from basic formulations such as phosphate-buffered saline (PBS), to advanced additive solutions (ASs), like AS-7 and phosphate, adenine, glucose, guanosine, saline, and mannitol (PAGGSM), are systematically compared in terms of their ability to maintain key indicators of RBC integrity, including adenosine triphosphate (ATP) levels, morphology, and hemolysis. Optimal RBC storage requires a delicate balance of pH buffering, metabolic support, oxidative damage prevention, and osmotic regulation. While the latest alkaline solutions enable up to 8 weeks of storage, some degree of metabolic and morphological deterioration remains inevitable. The impacts of critical storage conditions, such as the holding temperature, oxygenation, anticoagulants, irradiation, and processing methods, on the accumulation of storage lesions are also thoroughly investigated. Personalized RBC storage solutions, tailored to individual donor characteristics, represent a promising avenue for minimizing storage lesions and enhancing transfusion outcomes. Further research integrating omics profiling with customized preservation media is necessary to maximize post-transfusion RBC survival and functions. The continued optimization of RBC storage practices will not only enhance transfusion efficacy but also enable blood banking to better meet evolving clinical needs.

"Spatio-temporal symmetries of electronic chirality flips in oriented RbCs induced by two coincident laser pulses with circular ++,+-,-+,-- polarizations".

Recently it has been shown that two coincident well designed laser pulses with two different combinations of circular polarizations ( ++ or -+ ) can create chiral electronic densities in an oriented heteronuclear diatomic molecule. Subsequently, the chirality flips from the electronic Ra to Sa to Ra to Sa etc. enantiomers, with periods in the femtosecond (fs) and attosecond (as) time domains. The results were obtained by means of quantum dynamics simulations for oriented NaK. Here we investigate the electronic chirality flips in oriented RbCs induced by all possible ( ++ , -+ , +- , -- ) combinations of circular polarizations of two coincident well-designed laser pulses. Accordingly, the ++ and -- as well as the +- and -+ combinations generate opposite electronic enantiomers, e. g. Ra versus Sa, followed by opposite periodic chirality flips, e.g. form Ra to Sa to Ra to Sa  etc. versus form Sa to Ra to Sa to Ra  etc, with periods in the fs and as time domains, respectively. The laser induced spatio-temporal symmetries are derived from first principles and illustrated by quantum dynamics simulations.

Mitochondrial regulation of erythropoiesis in homeostasis and disease.

Erythroid cells undergo a highly complex maturation process, resulting in dynamic changes that generate red blood cells (RBCs) highly rich in haemoglobin. The end stages of the erythroid cell maturation process primarily include chromatin condensation and nuclear polarization, followed by nuclear expulsion called enucleation and clearance of mitochondria and other organelles to finally generate mature RBCs. While healthy RBCs are devoid of mitochondria, recent evidence suggests that mitochondria are actively implicated in the processes of erythroid cell maturation, erythroblast enucleation and RBC production. However, the extent of mitochondrial participation that occurs during these ultimate steps is not completely understood. This is specifically important since abnormal RBC retention of mitochondria or mitochondrial DNA contributes to the pathophysiology of sickle cell and other disorders. Here we review some of the key findings so far that elucidate the importance of this process in various aspects of erythroid maturation and RBC production under homeostasis and disease conditions.

Flexural strength and degree of conversion of universal single shade resin-based composites.

Background/purpose: Recently, a group of universal single-shade resin-based composites (RBCs) has been developed to simplify the process of shade selection. Excellent mechanical and physical properties are crucial for the ultimate success and clinical longevity of restorations. Therefore, evaluating the properties of the single-shaded RBCs is imperative. This study aimed to determine the flexural strength (FS) and degree of conversion (DC) of universal single-shade RBCs. Materials and methods: In this study, four commercial RBCs were used; three universal single-shade RBCs; Omnichroma (OC), Charisma® Diamond ONE (CD), and Vittra APS Unique (VU), and a conventional nanohybrid composite Filtek™ Z250 XT (FT) which was used as a control. Sixty composite beams and 40 composite discs were used for FS and DC, respectively. A universal test machine with a three-point bending test was used to measure the FS, whereas the DC was measured using a Fourier-transform infrared spectrometer (FTIR). Three fractured specimens from each resin composite group were qualitatively analyzed using scanning electron microscopy. Results: ANOVA was used to compare the mean values of FS and DC among the four RBCs (OC, CD, VU, and FT). Highly significant differences were observed in the mean FS and DC values (F = 673.043, p < 0.001 and F (=782.4, p < 0.0001), respectively. The highest FS was observed in the CD group, followed by FT and VU groups; the lowest value was observed in the OC group. In addition, a statistically significant difference was identified in DC values. The highest DC value was observed in VU, followed by OC and CD, and the lowest DC value was observed in FT. Conclusion: Universal single-shade RBCs demonstrated a good FS, except for OC, which exhibited a significantly low FS. The DC of the universal single-shade RBCs was higher than that of the conventional nanohybrid composite restorative material.

Unveiling the role of mtDNA in Liver-Kidney Crosstalk: Insights from trichloroethylene hypersensitivity syndrome.

In specific pathological conditions, addressing liver injury may yield favorable effects on renal function through the phenomenon of liver-kidney crosstalk. Mitochondrial DNA (mtDNA) possesses the capability to trigger downstream pathways of inflammatory cytokines, ultimately leading to immune-mediated organ damage. Consequently, understanding the intricate molecular mechanisms governing mtDNA involvement in diseases characterized by liver-kidney crosstalk is of paramount significance. This study seeks to elucidate the role of mtDNA in conditions marked by liver-kidney crosstalk. In previous clinical cases, it has been observed that patients with Trichloroethylene Hypersensitivity Syndrome (TCE-HS) who experience severe liver injury often also exhibit renal injury. In this study, patients diagnosed with trichloroethylene hypersensitivity syndrome were recruited from Shenzhen Occupational Disease Control Center. And Balb/c mice were treated with trichloroethylene. The correlation between liver and kidney injuries in patients with TCE-HS was assessed using Enzyme-Linked Immunosorbent Assay (ELISA). Alterations in mtDNA levels were examined in mouse hepatocytes, red blood cells (RBCs), and renal tubular epithelial cells utilizing immunofluorescence and PCR techniques. TCE-sensitized mice exhibited a significant increase in reactive oxygen species (ROS) and the opening of the mitochondrial permeability transition pore in hepatocytes, resulting in the release of mtDNA. Furthermore, heightened levels of mtDNA and Toll-like Receptor 9 (TLR9) expression were observed in RBCs. Additional experiments demonstrated elevated expression of TLR9 and its downstream mediator MyD88 in renal tubule epithelial cells of TCE-sensitized mice. In vitro investigations confirmed that mtDNA activates the TLR9 pathway in TCMK-1 cells. Collectively, these results suggest that mtDNA released from mitochondrial damage in hepatocytes is carried by RBCs to renal tubular epithelial cells and mediates inflammatory injury in renal tubular epithelial cells through activation of the TLR9 receptor.

Highly Defined Induced Pluripotent Stem Cell Lines Mimic Donor Red Blood Cell Antigen Profiles for Therapeutic and Diagnostic Use.

Our group generated two induced pluripotent stem cell (iPSC) lines for in vitro red blood cell (RBC) production from blood donors with extensively known erythrocyte antigen profiles. One line was intended to give rise to RBCs for transfusions in patients with sickle cell disease (SCD), while the other was developed to create RBC panel reagents. Two blood donors were selected based on their RBC phenotypes, further complemented by high-throughput DNA array analysis to obtain a more comprehensive erythrocyte antigen profile. Enriched erythroblast populations from the donors' peripheral blood mononuclear cells were reprogrammed into iPSCs using nonintegrative plasmid vectors. The iPSC lines were characterized and subsequently subjected to hematopoietic differentiation. iPSC PB02 and iPSC PB12 demonstrated in vitro and in vivo iPSC features and retained the genotype of each blood donor's RBC antigen profile. Colony-forming cell assays confirmed that iPSC PB02 and iPSC PB12 generated hematopoietic progenitors. These two iPSC lines were generated with defined erythrocyte antigen profiles, self-renewal capacity, and hematopoietic differentiation potential. With improvements in hematopoietic differentiation, these cells could potentially be more efficiently differentiated into RBCs in the future. They could serve as a complementary approach for obtaining donor-independent RBCs and addressing specific demands for blood transfusions.

Characterization of activating cis-regulatory elements from the histone genes of Chlamydomonas reinhardtii.

Regulation of gene expression in eukaryotes is controlled by cis-regulatory modules (CRMs). A major class of CRMs are enhancers which are composed of activating cis-regulatory elements (CREs) responsible for upregulating transcription. To date, most enhancers and activating CREs have been studied in angiosperms; in contrast, our knowledge about these key regulators of gene expression in green algae is limited. In this study, we aimed at characterizing putative activating CREs/CRMs from the histone genes of the unicellular model alga Chlamydomonas reinhardtii. To test the activity of four candidates, reporter constructs consisting of a tetramerized CRE, an established promoter, and a gene for the mCerulean3 fluorescent protein were incorporated into the nuclear genome of C. reinhardtii, and their activity was quantified by flow cytometry. Two tested candidates, Eupstr and Ehist cons, significantly upregulated gene expression and were characterized in detail. Eupstr, which originates from highly expressed genes of C. reinhardtii, is an orientation-independent CRE capable of activating both the RBCS2 and ß2-tubulin promoters. Ehist cons, which is a CRM from histone genes of angiosperms, upregulates the ß2-tubulin promoter in C. reinhardtii over a distance of at least 1.5 kb. The octamer motif present in Ehist cons was identified in C. reinhardtii and the related green algae Chlamydomonas incerta, Chlamydomonas schloesseri, and Edaphochlamys debaryana, demonstrating its high evolutionary conservation. The results of this investigation expand our knowledge about the regulation of gene expression in green algae. Furthermore, the characterized activating CREs/CRMs can be applied as valuable genetic tools.

Cerebral malaria pathogenesis: Dissecting the role of CD4+ and CD8+ T-cells as major effectors in disease pathology.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum (P. falciparum) infection, with complex pathogenesis involving multiple factors, including the host's immunological response. T lymphocytes, specifically CD4+ T helper cells and CD8+ cytotoxic T cells, are crucial in controlling parasite growth and activating cells for parasite clearance via cytokine secretion. Contrary to this, reports also suggest the pathogenic nature of T lymphocytes as they are often involved in disease progression and severity. CD8+ cytotoxic T cells migrate to the host's brain vasculature, disrupting the blood-brain barrier and causing neurological manifestations. CD4+ T helper cells on the other hand play a variety of functions as they differentiate into different subtypes which may function as pro-inflammatory or anti-inflammatory. The excessive pro-inflammatory response in CM can lead to multi-organ failure, necessitating a check mechanism to maintain immune homeostasis. This is achieved by regulatory T cells and their characteristic cytokines, which counterbalance the pro-inflammatory immune response. Maintaining a critical balance between pro and anti-inflammatory responses is crucial for determining disease outcomes in CM. A slight change in this balance may contribute to a disease severity owing to an extreme inflammatory response or unrestricted parasite growth, a potential target for designing immunotherapeutic treatment approaches. The review briefly discusses the pathogenesis of CM and various mechanisms responsible for the disruption of the blood-brain barrier. It also highlights the role of different T cell subsets during infection and emphasizes the importance of balance between pro and anti-inflammatory T cells that ultimately decides the outcome of the disease.

CM is potentially fatal complication of P. falciparum infection that presents with high mortality and morbidity. Vaccines are extensively being developed against the Plasmodium parasite but very few of them are effective. Artemisinin Combination Therapy (ACT) is a major treatment for malaria, but its effectiveness is declining due to Plasmodium sp. developing resistance to it, necessitating the need for development of new drugs and treatments. During infection, the parasite is responsible for causing infected red blood cell (RBC) sequestration and cytoadherence in brain vasculature and extreme pro-inflammatory response that ultimately causes endothelial dysfunction and bloodbrain barrier (BBB) disruption. The host initiates a pro-inflammatory response against the parasite which includes activation of cells of both innate and adaptive immune response. These cells control the parasite growth and aid in parasite clearance from host's body. The inflammatory response generally targets foreign pathogens and provides protection against possible infection but can also cause harm to the self when left unchecked. It has been reported that activated immune cells, mainly T-lymphocytes often migrate to brain vasculature and ultimately results in neuronal damage characteristic CM. To counteract the overwhelming pro-inflammatory response, the host immune system deploys an anti-inflammatory response, which often involves regulatory cells and cytokines that help the body maintain immunological homeostasis. The review briefly highlights the necessity of balancing the pro- and anti-inflammatory responses for successful parasite clearance without the deleterious effects to the host that might increase disease severity in CM.

Erythrocytes Nanoparticle Delivery: A Boon for Targeting Tumor.

Although nanoparticles (NPs) have many advantages as drug delivery systems, their poor stability in circulation, premature drug release, and nonspecific uptake in non-target organs have prompted biomimetic approaches to camouflage nano vehicles using natural cell membranes. Among them, which are extensively studied in erythrocytes, are the most abundant circulating blood cells. They are specially used for biomimetic coating on artificial NPs due to their excellent properties of good biocompatibility, biodegradability, non-immunogenicity, and long-term blood circulation. Erythrocyte-mimicking nanoparticles (EM-NPs) are prepared by combining nanoparticle cores with naturally derived erythrocyte (red blood cell or RBC) membranes. Compared with conventional nanosystems, EM-NPs hold the preferable characteristics of prolonged blood circulation time and immune evasion. In this review, the biomimetic platform of erythrocyte membrane-coated NPs is described in various aspects, with particular focus placed on the coating mechanism, preparation methods, characterization method, and recent advances in the biomedical applications of EM-NPs concerning cancer and targeted delivery.

Reference intervals of complete blood count parameters in the adult western Sudanese population.

BACKGROUND: A complete blood count (CBC) analysis is one of the most common conventional blood tests that physicians frequently prescribe. THE OBJECTIVE: of this study was to determine the reference intervals (RIs) of CBC parameters in the population of healthy adults living in the western Sudan region. METHODS: A cross-sectional study of healthy people residing in the western area of Sudan was carried out. We assessed the CBC RIs in samples taken from 153 individuals using an automated haematology analyser (Sysmex KX-21) and a modified Box-Cox transformation procedure to transform the data into a Gaussian distribution after eliminating outliers using the Dixon method. IBM SPSS Statistics version 25 was used to analyse the data, and t tests were employed to examine variations in the mean CBC parameters according to sex and age. P was considered significant at ≤ 0.05. RESULTS: Beyond all the other measured values, the only CBC parameters that significantly differed between the sexes were haemoglobin (HGB) and white blood cell (WBC) counts. Women were found to experience more WBC counts than men did. However, they have less HGB RIs.The male participants in our study exhibited lower WBC count RIs, a significantly lower limit, and a greater upper limit of platelet RIs than did the individuals from other nations. CONCLUSIONS: Compared with males, females had higher platelet and WBC counts and lower HGB.

Antimalarial Effects of Nano Chloroquine Loaded Curcumin In vivo.

BACKGROUND: Malaria is still the deadliest parasitic disease caused by Plasmodium spp. Due to drug resistance and their unpleasant side effects, of conventional researchers are enormously seeking to achieve antimalarial drugs with more curative effective, less toxic and cost-affordable drugs using more advanced technology such as nanodrugs. PURPOSE: The present study aimed to examine the antimalarial effects of a novel synthesized nonochloroquine-loaded curcumin relying on dendrimer G2 in susceptible mice. METHODS: Antimalarial activity and toxicity of the nanocomposite were examined on BALB/C mice with microscopy, checking RBCs morphology and related enzymatic activity rate. RESULTS: The maximum inhibitory effect of the nanocomposite was seen at 10 mg/kg, killing 98% of P. berghei compared to sole chloroquine, whereas ED50 was reported at 5.5 mg/kg. The safety of the synthesized nanocomposite was confirmed with biochemical tests with no detrimental effects on mice. The sustainability and longevity of the nanodrug increased significantly with the NDC-CQ assay compared to the control groups. CONCLUSION: The study showed that nonochloroquine-loaded curcumin had a promising inhibitory effect on P. berghei growth in infected mice compared to standard drugs. However, further studies and clinical trials with large samples are recommended to study different aspects of using nanodrug.

Human erythrocytes under stress. Spectroscopic fingerprints of known oxidative mechanisms and beyond.

In this work, we investigated the oxidative stress-related biochemical alterations in red blood cells (RBCs) and their membranes with the use of spectroscopic techniques. We aimed to show their great advantage for the in situ detection of lipid classes and secondary structures of proteins without the need for their extraction in the cellular environment. The exposition of the cells to peroxides, t-butyl hydroperoxide (tBOOH) or hydrogen peroxide (H2O2) led to different degradation processes encompassing the changes in the composition of membranes and structural modifications of hemoglobin (Hb). Our results indicated that tBOOH is generally a stronger oxidizing agent than H2O2 and this observation was congruent with the activity of superoxide and glutathione peroxidase. ATR-FTIR and Raman spectroscopies of membranes revealed that tBOOH caused primarily the partial loss and peroxidation of the lipids resulting in loss of the integrity of membranes. In turn, both peroxides induced several kinds of damage in the protein layer, including the partial decrease of their content and irreversible aggregation of spectrin, ankyrin, and membrane-bound globin. These changes were especially pronounced on the membrane surface where stress conditions induced the formation of ß-sheets and intramolecular aggregates, particularly for tBOOH. Interestingly, nano-FTIR spectroscopy revealed the lipid peroxidative damage on the membrane surface in both cases. As far as hemoglobin was concerned, tBOOH and H2O2 caused the increase of the oxyhemoglobin species and conformational alterations of its polypeptide chain into ß-sheets. Our findings confirm that applied spectroscopies effectively track the oxidative changes occurring in the structural components of red blood cells and the simplicity of conducting measurements and sample preparation can be readily applied to pharmacological and clinical studies.

Effect of different finishing and polishing systems on surface properties of universal single shade resin-based composites.

BACKGROUND: Recently, universal single-shade resin composites have become increasingly available in the dental market. The modification of their composition can have an inadvertent effect on their physical and surface properties, and subsequently determinantal effect on their clinical function and longevity. Therefore, this study aimed to evaluate the effect of different finishing and polishing (F/P) systems on surface roughness (Ra), surface gloss (GU), and Vickers microhardness (VMH) of universal single-shade RBCs. MATERIALS AND METHODS: Four commercial RBCs were used; the universal single-shade RBCs were Omnichroma, Charisma® Diamond ONE, and Vittra APS Unique, and a conventional nanocomposite Filtek™ Z250 XT was used as a control. The 3 F/P systems were Sof-Lex™ XT, Enhance®/PoGo®, and Diacomp® Plus Twist. A total of 160 discs were used for the 3 F/P system groups for all RBCs (n = 10). After F/P, the Ra, GU, and VMH were assessed. The data were analyzed using 2-way ANOVA at p-value < 0.05. RESULTS: Significant differences were found among the four RBCs and the 3 F/P systems (p < .000). Omnichroma showed the lowest Ra and acceptable GU, but the lowest VMH. Charisma showed the highest Ra, acceptable GU, and VMH. Vittra showed acceptable Ra, GU, and VMH and Filtek showed the highest GU, VMH, and acceptable Ra. CONCLUSION: Although conventional nanohybrid RBC (Filtek Z250 XT) showed better GU and VMH values, the universal single-shade RBCs demonstrated comparable surface properties. The highest GU & VMH and lowest Ra were achieved by Diacomp followed by Enhance and Soflex.

Drug-loaded erythrocytes: Modern approaches for advanced drug delivery for clinical use.

Scientific organizations worldwide are striving to create drug delivery systems that provide a high local concentration of a drug in pathological tissue without side effects on healthy organs in the body. Important physiological properties of red blood cells (RBCs), such as frequent renewal ability, good oxygen carrying ability, unique shape and membrane flexibility, allow them to be used as natural carriers of drugs in the body. Erythrocyte carriers derived from autologous blood are even more promising drug delivery systems due to their immunogenic compatibility, safety, natural uniqueness, simple preparation, biodegradability and convenience of use in clinical practice. This review is focused on the achievements in the clinical application of targeted drug delivery systems based on osmotic methods of loading RBCs, with an emphasis on advancements in their industrial production. This article describes the basic methods used for encapsulating drugs into erythrocytes, key strategic approaches to the clinical use of drug-loaded erythrocytes obtained by hypotonic hemolysis. Moreover, clinical trials of erythrocyte carriers for the targeted delivery are discussed. This article explores the recent advancements and engineering approaches employed in the encapsulation of erythrocytes through hypotonic hemolysis methods, as well as the most promising inventions in this field. There is currently a shortage of reviews focused on the automation of drug loading into RBCs; therefore, our work fills this gap. Finally, further prospects for the development of engineering and technological solutions for the automatic production of drug-loaded RBCs were studied. Automated devices have the potential to provide the widespread production of RBC-encapsulated therapeutic drugs and optimize the process of targeted drug delivery in the body. Furthermore, they can expedite the widespread introduction of this innovative treatment method into clinical practice, thereby significantly expanding the effectiveness of treatment in both surgery and all areas of medicine.

Light-sheet Raman tweezers for whole-cell biochemical analysis of functional red blood cells.

Micro-Raman spectroscopy has emerged as one of the foremost techniques for analyzing biological cells in recent years due to its non-destructive nature and high spatial resolution. The development of optical tweezers has eased the research on biological cells as they confine living cells and organisms in the optical trap without causing much damage. Combining optical tweezers with Raman spectroscopy has opened a wide range of applications in the biomedical field as it facilitates biochemical analysis of biological samples by maintaining in-vivo conditions. Herein, we developed a light sheet-based optical tweezer that traps red blood cells (RBCs) at a very low power density spread across the whole cell, otherwise impossible with conventional optical tweezers. Furthermore, it is combined with micro-Raman spectroscopy to perform whole-cell biochemical analysis for the first time. Raman spectra of individual RBCs recorded under the line focal spot excitation are of superior quality and lack spectral signatures of photo-oxidation and heme aggregation, which is common in point focal spot excitations.

Hypoxically stored RBC resuscitation in a rat model of traumatic brain injury and severe hemorrhagic shock.

This study aims to investigate the effects of hypoxically stored Red Blood Cells (RBCs) in a rat model of traumatic brain injury followed by severe hemorrhagic shock (HS) and resuscitation. RBCs were made hypoxic using an O2 depletion system (Hemanext Inc. Lexington, MA) and stored for 3 weeks. Experimental animals underwent craniotomy and blunt brain injury followed by severe HS. Rats were resuscitated with either fresh RBCs (FRBCs), 3-week-old hypoxically stored RBCs (HRBCs), or 3-week-old conventionally stored RBCs (CRBCs). Resuscitation was provided via RBCs transfusion equivalent to 70 % of the shed blood and animals were followed for 2 h. The control group was comprised of healthy animals that were not instrumented or injured. Post-resuscitation hemodynamics and lactate levels were improved with FRBCs and HRBCs, and markers of organ injury in the liver (Aspartate aminotransferase [AST]), lung (chemokine ligand 1 [CXCL-1] and Leukocytes count), and heart (cardiac troponin, Interleukin- 6 [IL-6] and Tumor Necrosis Factor Alpha[TNF-α]) were lower with FRBCs and HRBCs resuscitation compared to CRBCs. Following reperfusion, biomarkers for oxidative stress, lipid peroxidation, and RNA/DNA injury were assessed. Superoxide dismutase [SOD] levels in the HRBCs group were similar to the FRBCs group and levels in both groups were significantly higher than CRBCs. Catalase levels were not different than control values in the FRBCs and HRBCs groups but significantly lower with CRBCs. Thiobarbituric acid reactive substances [Tbars] levels were higher for both CRBCs and HRBCs. Hypoxically stored RBCs show few differences from fresh RBCs in resuscitation from TBI + HS and decreased organ injury and oxidative stress compared to conventionally stored RBCs.

Nanoengineered Red Blood Cells Loaded with TMPRSS2 and Cathepsin L Inhibitors Block SARS-CoV-2 Pseudovirus Entry into Lung ACE2+ Cells.

The enzymatic activities of Furin, Transmembrane serine proteinase 2 (TMPRSS2), Cathepsin L (CTSL), and Angiotensin-converting enzyme 2 (ACE2) receptor binding are necessary for the entry of coronaviruses into host cells. Precise inhibition of these key proteases in ACE2+ lung cells during a viral infection cycle shall prevent viral Spike (S) protein activation and its fusion with a host cell membrane, consequently averting virus entry to the cells. In this study, dual-drug-combined (TMPRSS2 inhibitor Camostat and CTSL inhibitor E-64d) nanocarriers (NCs) are constructed conjugated with an anti-human ACE2 (hACE2) antibody and employ Red Blood Cell (RBC)-hitchhiking, termed "Nanoengineered RBCs," for targeting lung cells. The significant therapeutic efficacy of the dual-drug-loaded nanoengineered RBCs in pseudovirus-infected K18-hACE2 transgenic mice is reported. Notably, the modular nanoengineered RBCs (anti-receptor antibody+NCs+RBCs) precisely target key proteases of host cells in the lungs to block the entry of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), regardless of virus variations. These findings are anticipated to benefit the development of a series of novel and safe host-cell-protecting antiviral therapies.

Organogermanium, Ge-132, promotes the clearance of senescent red blood cells via macrophage-mediated phagocyte activation.

Red blood cells (RBCs) are renewed in a cyclic manner. Aging RBCs are captured and degraded by phagocytic cells, and heme metabolic pigments are subsequently excreted in feces. We evaluated the effect of an organogermanium compound on RBC metabolism and found that the phagocytosis of RAW264.7 macrophage-like cells was increased by treatment with 3-(trihydroxygermyl)propanoic acid (THGP). Additionally, consumption of Ge-132 (a dehydrate polymer of THGP) changed the fecal color to bright yellow and increased the erythrocyte metabolic pigment levels and antioxidant activity in feces. These data suggest that Ge-132 may activate macrophages in the body and promote the degradation of aged RBCs. Furthermore, Ge-132 intake promoted not only increases in RBC degradation but also the induction of erythroblast differentiation in bone marrow cells. The normal hematocrit levels were maintained due to the maintenance of homeostasis, even though Ge-132 ingestion increased erythrocyte degradation. Therefore, Ge-132 enhances the degradation of senescent RBCs by macrophages. In turn, RBC production is increased to compensate for the amount of degradation, and RBC metabolism is increased.