PHE1-based IgG-like antibody platform provides a novel strategy for enhanced T-cell immunotherapy.

Introduction: Bispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets, bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody, which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time. Methods: This study comprised antibodies purification, Elisa assay for antigen binding, cytotoxicity assays, T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo. Results: We present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin ß2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially, BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo. Discussion: Moreover, PHE1 domain was further shortened with D14G and R41S mutations, named PHE-S, and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo, bringing more possibilities for the development and optimization of different bsAbs. To sum up, PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy.

The role of PALLD-STAT3 interaction in megakaryocyte differentiation and thrombocytopenia treatment.

Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a crucial transcription factor in regulating megakaryocyte differentiation, yet the precise mechanism of its activation remains unclear. PALLD, an actin-associated protein, has been increasingly recognized for its essential functions in multiple biological processes. This study revealed that megakaryocyte/plateletspecific knockout of PALLD in mice exhibited thrombocytopenia due to diminished platelet biogenesis. In megakaryocytes, PALLD deficiency led to impaired proplatelet formation and polyploidization, ultimately weakening their differentiation for platelet production. Mechanistic studies demonstrated that PALLD bound to STAT3 and interacted with its DNA-binding domain (DBD) and Src homology 2 (SH2) domain via Immunoglobulin domain 3 (Ig3). Moreover, the absence of PALLD attenuated STAT3 Y705 phosphorylation and impeded STAT3 nuclear translocation. Based on the PALLD-STAT3 binding sequence, we designed a peptide C-P3, which can facilitate megakaryocyte differentiation and accelerate platelet production in vivo. In conclusion, this study highlights the pivotal role of PALLD in megakaryocyte differentiation and proposes a novel approach for treating thrombocytopenia by targeting the PALLD-STAT3 interaction.

Whether the Unilateral Transverse Process-pedicle Approach has Advantages over the Traditional Transpedicle Approach: A Systematic review and Meta-analysis.

PURPOSE: To summarize the literature and compare the advantages and disadvantages of the unilateral transverse process-pedicle approach (UTPA) and conventional transpedicular approach (CTPA) vertebral augmentation in the treatment of osteoporotic vertebral compression fractures (OVCF). METHODS: A single researcher performed a systematic literature review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Online scientific databases were searched in September 2021 for English- and Chinese-language publications. A series of comparative studies were included, with UTPA as the main intervention and CTPA as the comparison indicator. A meta-analysis was performed for studies that reported clinical outcome indicators. The χ2 was used to study heterogeneity between trials, and the I2 statistic was calculated to estimate variation across studies. RESULTS: A total of eight studies were included for meta-analysis, all of which were observational studies with mixed bias risk. There were 613 subjects in the UTPA group and 488 subjects in the CTPA group. The results of the meta-analysis showed that there was no difference between the UTPA group and the CTPA group in terms of visual analogue scale scores (p = 0.31), Oswestry Disability Index scores (p = 0.50), correction of kyphosis angle (p = 0.65), and the amount of bone cement (p = 0.13), but the UTPA group had a shorter operative time (p < 0.001), bone cement leakage rates (p = 0.02), and fluoroscopy times than the CTPA group (p < 0.001). Partial analysis results had a high risk of bias, and the most common source of bias was that there was high heterogeneity between studies, and the sensitivity can only be reduced by a random effect model, and some studies (four items) did not clearly describe the confounders that they controlled. CONCLUSION: The limited evidence obtained in this study proves that the new puncture method does not have more advantages than the traditional technique, so it is no longer meaningful to continue to obsess over the impact of the puncture method on surgical outcome.

Isoquercitrin promotes peripheral nerve regeneration through inhibiting oxidative stress following sciatic crush injury in mice.

BACKGROUND: Oxidative stress has been recognized to play a crucial role in the pathogenesis of peripheral nerve injury. Isoquercitrin (quercetin-3-glucoside) is a flavonoid that exhibited many biological activities, including anti-oxidative effect. However, it is unclear whether isoquercitrin has protective effects on peripheral nerve injury. METHODS: Mice treated by isoquercitrin were used as a case group, and mice injected with saline was the control group. Sciatic behavioral function was assessed using SFI and CMAPs were measured by electrophysiology. Schwann cells proliferation and migration were tested using EdU staining and Transwell migration chambers respectively. The expression of oxidative stress related factors were detected by qRT-PCR and Western blotting. RESULTS: In present study, our results demonstrated that isoquercitrin (20 mg/kg/day) treatment achieved significantly higher SFI and higher amplitude of CMAP, promoted the nerve regeneration and remyelination, increased the production of GAP43, NF200, MAG and PMP22, alleviated target muscle atrophy and autophagy, and suppressed the expression of ATG7, PINK1 and Beclin1 in soleus muscles after sciatic nerve crush. In vitro studies found that isoquercitrin promoted the axonal regeneration of DRGs neurons, the proliferation and migration of Schwann cells, and the expression of proliferating cell nuclear antigen (PCNA) in Schwann cells. The administration of isoquercitrin at 40 and 320 µM showed a dose dependent, and high doses of isoquercitrin (160 and 320 µM) showed better performance in promoting axonal regeneration of DRGs neurons, and the proliferation and migration of Schwann cells than low dose of isoquercitrin (40 µM). Furthermore, isoquercitrin significantly inhibited oxidative stress through reducing the production of Nox4 and Duox1, and promoting the expression of Nrf2 and SOD2 in soleus muscles after sciatic nerve crush. CONCLUSIONS: Isoquercitrin may promote motor functional recovery and nerve regeneration following peripheral nerve injury though inhibition of oxidative stress, which highlighted the therapeutic values of isoquercitrin as a neuroprotective drug for peripheral nerve repair applications.

Pyrroloquinoline quinone attenuates cachexia-induced muscle atrophy via suppression of reactive oxygen species.

BACKGROUND: Cachexia, a wasting syndrome, is most commonly observed in individuals with advanced cancer including lung cancer, esophageal cancer, liver cancer, etc. The characteristic sign of cachexia is muscle atrophy. To date, effective countermeasures have been still deficiency to alleviate muscle atrophy. Reactive oxygen species (ROS) are important regulators of muscle atrophy. Therefore, the effects of a naturally antioxidant, pyrroloquinoline quinone (PQQ), were explored on muscle atrophy induced by cachexia in the present study. METHODS: Tumor necrosis factor-α (TNF-α) induced C2C12 myotubes atrophy model was constructed. The atrophied C2C12 myotubes were dealt with the presence or absence of N-acetyl-L-cysteine (NAC, an antioxidant for ROS abolition) (5 mM) or PQQ (80 µM) for 24 hours. ROS content was determined by dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The diameter of myotubes was analyzed by myosin heavy chain (MHC) staining. The protein levels of MHC, muscle atrophy F-box (MAFbx) and muscle RING finger-1 (MuRF-1) in each group were observed by Western blotting. RESULTS: First, ROS generation was enhanced in C2C12 myotubes treated with TNF-α. NAC treatments significantly avoided the reduction in the diameter of C2C12 myotubes, and concomitantly increased MHC levels, and decreased ROS contents, MuRF1 and MAFbx levels. These data suggested that the increased ROS induced by TNF-α might play a central role in muscle wasting. PQQ (a naturally occurring antioxidant) administration inhibited C2C12 myotubes atrophy induced by TNF-α, as evidenced by the increase of the diameter of C2C12 myotubes, together with increased MHC levels and decreased ROS, MAFbx and MuRF-1 levels. CONCLUSIONS: PQQ resists atrophic effect dependent on, at least in part, decreased ROS in skeletal muscle treated with TNF-α.

Mechanistic Role of Reactive Oxygen Species and Therapeutic Potential of Antioxidants in Denervation- or Fasting-Induced Skeletal Muscle Atrophy.

Skeletal muscle atrophy occurs under various conditions, such as disuse, denervation, fasting, aging, and various diseases. Although the underlying molecular mechanisms are still not fully understood, skeletal muscle atrophy is closely associated with reactive oxygen species (ROS) overproduction. In this study, we aimed to investigate the involvement of ROS in skeletal muscle atrophy from the perspective of gene regulation, and further examine therapeutic effects of antioxidants on skeletal muscle atrophy. Microarray data showed that the gene expression of many positive regulators for ROS production were up-regulated and the gene expression of many negative regulators for ROS production were down-regulated in mouse soleus muscle atrophied by denervation (sciatic nerve injury). The ROS level was significantly increased in denervated mouse soleus muscle or fasted C2C12 myotubes that had suffered from fasting (nutrient deprivation). These two muscle samples were then treated with N-acetyl-L-cysteine (NAC, a clinically used antioxidant) or pyrroloquinoline quinone (PQQ, a naturally occurring antioxidant), respectively. As compared to non-treatment, both NAC and PQQ treatment (1) reversed the increase in the ROS level in two muscle samples; (2) attenuated the reduction in the cross-sectional area (CSA) of denervated mouse muscle or in the diameter of fasted C2C12 myotube; (3) increased the myosin heavy chain (MHC) level and decreased the muscle atrophy F-box (MAFbx) and muscle-specific RING finger-1 (MuRF-1) levels in two muscle samples. Collectively, these results suggested that an increased ROS level was, at least partly, responsible for denervation- or fasting-induced skeletal muscle atrophy, and antioxidants might resist the atrophic effect via ROS-related mechanisms.

MicroRNA351 targeting TRAF6 alleviates dexamethasone-induced myotube atrophy.

BACKGROUND: Glucocorticoids, including dexamethasone (Dex), are corticosteroids secreted by the adrenal gland, which are used as potent anti-inflammatory, anti-shock, and immunosuppressive agents. Dex is commonly used in patients with malignant tumors, such lung cancer. However, administration of high-dose Dex induces severe atrophy of the skeletal muscle, and the underlying mechanisms of this skeletal muscle atrophy remain unclear. Abundant miRNAs of skeletal muscle, such as miR-351, play an important role in the regulation of extenuating the process of muscle atrophy. METHODS: The mRNA and protein expression of TRAF6, MuRF1, MAFbx was determined by real-time PCR and western blot, while the expression of miR-351 was detected by real-time PCR. The myotubes were transfected with miR-351 mimic, negative control, or miR-351 inhibitor. The C2C12 myotubes diameter was measured. RESULTS: MicroRNA351 (miR-351) level was markedly reduced and the mRNA and protein levels of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) were increased in Dex-induced C2C12 myotube atrophy. miR-351 directly interacted with the 3'-untranslated region (3'UTR) of TRAF6. Interestingly, miR-351 administration notably inhibited the reduction of the C2C12 myotube diameter induced by Dex treatment and reduced the levels of TRAF6, muscle-RING-finger protein-1 (MuRF1), and muscle atrophy F-box (MAFbx). CONCLUSIONS: miR-351 counteracts Dex-induced C2C12 myotube atrophy by repressing the TRAF6 expression as well as E3 ubiquitin ligase MuRF1 and MAFbx. miR-351 maybe a potential target for development of a new strategy for skeletal muscle atrophy.

Analysis of algae growth mechanism and water bloom prediction under the effect of multi-affecting factor.

The formation process of algae is described inaccurately and water blooms are predicted with a low precision by current methods. In this paper, chemical mechanism of algae growth is analyzed, and a correlation analysis of chlorophyll-a and algal density is conducted by chemical measurement. Taking into account the influence of multi-factors on algae growth and water blooms, the comprehensive prediction method combined with multivariate time series and intelligent model is put forward in this paper. Firstly, through the process of photosynthesis, the main factors that affect the reproduction of the algae are analyzed. A compensation prediction method of multivariate time series analysis based on neural network and Support Vector Machine has been put forward which is combined with Kernel Principal Component Analysis to deal with dimension reduction of the influence factors of blooms. Then, Genetic Algorithm is applied to improve the generalization ability of the BP network and Least Squares Support Vector Machine. Experimental results show that this method could better compensate the prediction model of multivariate time series analysis which is an effective way to improve the description accuracy of algae growth and prediction precision of water blooms.

Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer.

Sperm-mediated gene transfer (SMGT) has been long heralded as a faster and cheaper alternative to more commonly used methods of producing transgenic animals. In this study, the capra semen ejaculates were pooled together and then incubated in vitro with DIG-labeled DNA. The binding and internalizing rates were observed by the in situ hybridization methods. We also compared the standard sperm parameters and the efficiencies of interaction with exogenous DNA of 60 individuals to select donor bucks for SMGT. It was showed that labeled exogenous DNA was detected in different localizations in spermatozoa but genuine DNA uptake, in contrast to mere binding, seems to be limited to the postacrosomal region. The removal of seminal plasma increased significantly (P < 0.01) the capability in picking up exogenous DNA. Use of frozen-thawed semen (without cryoprotectant agents) and Triton X-100 treatment also increased significantly (P < 0.01) the DNA-binding capacity, but reduced the sperm viability. The binding rates (the proportion of labeled-DNA positive spermatozoa to all the spermatozoa counted) of 60 buck individuals were in the range of 3.08-73.39%, and the internalizing rates (the proportion of DNaseI-treated labeled-DNA positive spermatozoa to all the spermatozoa counted) were 4.83-70.00%. About 8.34% (5/60) bucks showed high binding, but low internalizing ability. Chi-square test showed that there was significant difference among the breeds (x(2) = 26.515, P < 0.01). Eight individual bucks that demonstrated high DNA uptake were selected for SMGT. It was demonstrated that the goat spermatozoa was capable of spontaneous uptake of exogenous DNA. Seminal fluid inhibits DNA uptake and that membrane disruption increases DNA binding but greatly diminishes uptake.

Association of goat (Capra hircus) CD4 gene exon 6 polymorphisms with ability of sperm internalizing exogenous DNA.

As one of the transport systems on the sperm plasma membrane, CD4 molecule plays a distinct role in the process of sperm/DNA interaction. This makes it possible to explain the mechanism of sperm-mediated gene transfer (SMGT), which at present is still a mystery in this area. In this study, seminal samples of 60 individuals from seven breed bucks were collected to detect the ability of sperm in internalizing exogenous DNA, and genomic DNA from 147 individual blood samples (including 60 bucks referred ahead) were extracted to test the polymorphisms of CD4 genes by using PCR-SSCP technique. Then the correlation between them was evaluated. The results showed that: (1) it was a novel finding that breed-dependence of exogenous DNA binding to goat spermatozoa. There was the most significant difference among the buck breeds of sperm in binding exogenous DNA (F((6, 53)) = 4.811, P = 0.001) and in internalizing them into nuclei (F((6, 53)) = 4.587, P = 0.001). The ability of Lezhi Black goat was the lowest (P < 0.01) among the seven breeds. (2) There was no significant correlation between the ability of sperm in internalizing exogenous DNA and each semen quality parameter (P > 0.05). (3) In particular, three single-nucleotide polymorphisms (SNP) were described and there was one SNP (G/A(700)) of CD4 gene that made G234R substitution in the amino acid sequence of CD4 molecule. Nanjiang Yellow goat and Lezhi Black goat had higher hereditary variation compared with other breeds. (4) CD4 polymorphisms were highly associated with the ability of sperm in internalizing exogenous DNA. The SNP of Caprine CD4 gene exon 6 might be an important molecular marker of the ability to internalize exogenous DNA into sperm.