Two new species of the order Monhysterida (Nematoda) from the sea of China.

Two new marine nematode species belonging to the order Monhysterida are described from the sea of China. Halomonhysterazhangisp. nov. is characterized by its relatively small body size; short anterior sensory setae; small, circular amphidial fovea located at the level of buccal cavity base; funnel-shaped buccal cavity; an excretory-secretory system with a large ventral gland and opening as a very narrow canal just posterior to the level of cephalic setae; slightly curved slender spicules with cephalated proximal end and tapered distal end; rod-like gubernaculum without apophysis; two papilliform precloacal supplements just in front of the cloaca; conico-cylindrical tail with two ventral papillae, each with a seta; and distance between the vulva and anus slightly longer than the tail length. This is the first new species of epiphytic nematode reported in China. The second new species, Stylotheristusflagellicaudatussp. nov., has a relatively shorter body and longer tail; striated cuticle; The anterior sensilla arranged in two circles, the first circle consisting of six inner labial setiform papillae (3-4 µm) and the second circle consisting of 16 long setae (12-19 µm); a transversely oval amphideal fovea; a well-developed muscle around the funnel-shaped buccal cavity; short spicules and a gubernaculum composed of a single piece; and precloacal supplements absent. An updated key to all species of Halomonhystera and pictorial key to all species of Stylotheristus are also given.

Two new species of Promonhystera Wieser, 1956 (Nematoda: Monhysterida: Xyalidae) from the South China Sea.

Two new species of free-living marine nematodes from the littoral sediment of the South China Sea are described as Promonhystera papilla sp. nov. and Promonhystera fluctispicula sp. nov. Both new species are characterized by striated cuticle; circular amphideal fovea; anterior sensilla in two circles, inner labial sensilla setiform; funnel-shaped buccal cavity; elongated spicules and conico-cylindrical tail. P. papilla sp. nov. is different from all known species of the genus by longer body length (more than 2.1mm versus less than 1.5 mm in other species), longer spicules (sp/a.b.d. = 4.14.6 versus 2.7) and males with three papilliform precloacal supplements and one small postcloacal papilla (versus absence) and gubernaculum boot-shaped. P. fluctispicula sp. nov. is similar to P. tricuspidata Wieser, 1956 in having similar waved spicules but differs from the latter species by tail conico-cylindrical (versus conical) and simple plate-like gubernaculum without tooth (versus gubernaculum with four distal teeth).

Development of curcumin-loaded galactosylated chitosan-coated nanoparticles for targeted delivery of hepatocellular carcinoma.

Curcumin (CUR) has good antitumor effects, but its poor aqueous solubility severely limits its clinical application and the systemic nonspecific distribution of the free drug in tumor patients is a key therapeutic challenge. In order to overcome the limitations of free drugs and improve the therapeutic efficacy, we developed novel galactosylated chitosan (GC)-modified nanoparticles (GC@NPs) based on poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) (PEG-PLGA), which can target asialoglycoprotein receptor (ASGPR) expressed on hepatocellular carcinoma cells and have excellent biocompatibility. The results showed that the drug loading (DL) of CUR was approximately 4.56 %. A favorable biosafety profile was maintained up to concentrations of 500 µg/mL. Furthermore, in vitro cellular assays showed that GC@NPs could be efficiently internalized by HepG2 cells via ASGPR-mediated endocytosis and successfully released CUR for chemotherapy. More importantly, in vivo anti-tumor experiments revealed that GC@NPs were able to accumulate effectively within tumor sites through EPR effect and ASGPR-mediated endocytosis, leading to superior inhibition of tumor growth compared to free CUR. Overall, GC@NPs are a promising CUR nanocarrier for enhanced tumor therapy with a good biosafety profile.

Targeted Drug Delivery Systems for Curcumin in Breast Cancer Therapy.

Breast cancer (BC) is the most prevalent type of cancer in the world and the main reason women die from cancer. Due to the significant side effects of conventional treatments such as chemotherapy and radiotherapy, the search for supplemental and alternative natural drugs with lower toxicity and side effects is of interest to researchers. Curcumin (CUR) is a natural polyphenol extracted from turmeric. Numerous studies have demonstrated that CUR is an effective anticancer drug that works by modifying different intracellular signaling pathways. CUR's therapeutic utility is severely constrained by its short half-life in vivo, low water solubility, poor stability, quick metabolism, low oral bioavailability, and potential for gastrointestinal discomfort with high oral doses. One of the most practical solutions to the aforementioned issues is the development of targeted drug delivery systems (TDDSs) based on nanomaterials. To improve drug targeting and efficacy and to serve as a reference for the development and use of CUR TDDSs in the clinical setting, this review describes the physicochemical properties and bioavailability of CUR and its mechanism of action on BC, with emphasis on recent studies on TDDSs for BC in combination with CUR, including passive TDDSs, active TDDSs and physicochemical TDDSs.

On-bead purification and nanodisc reconstitution of human chemokine receptor complexes for structural and biophysical studies.

Chemokine receptors, a subfamily of G-protein-coupled receptors (GPCRs), are responsible for cell migration during physiological processes as well as in diseases like inflammation and cancers. Here, we present a protocol for solubilizing, purifying, and reconstituting complexes of chemokine receptors with their ligands in "nanodiscs," soluble lipid bilayers that mimic the native environment of membrane receptors. The protocol yields chemokine receptor complexes with sufficient purity and yield for structural and biophysical studies and should be applicable to other GPCRs.

Development, Characterization, and Structural Analysis of a Genetically Encoded Red Fluorescent Peroxynitrite Biosensor.

Boronic acid-containing fluorescent molecules have been widely used to sense hydrogen peroxide and peroxynitrite, which are important reactive oxygen and nitrogen species in biological systems. However, it has been challenging to gain specificity. Our previous studies developed genetically encoded, green fluorescent peroxynitrite biosensors by genetically incorporating a boronic acid-containing noncanonical amino acid (ncAA), p-boronophenylalanine (pBoF), into the chromophore of circularly permuted green fluorescent proteins (cpGFPs). In this work, we introduced pBoF to amino acid residues spatially close to the chromophore of an enhanced circularly permuted red fluorescent protein (ecpApple). Our effort has resulted in two responsive ecpApple mutants: one bestows reactivity toward both peroxynitrite and hydrogen peroxide, while the other, namely, pnRFP, is a selective red fluorescent peroxynitrite biosensor. We characterized pnRFP in vitro and in live mammalian cells. We further studied the structure and sensing mechanism of pnRFP using X-ray crystallography, 11B-NMR, and computational methods. The boron atom in pnRFP adopts an sp2-hybridization geometry in a hydrophobic pocket, and the reaction of pnRFP with peroxynitrite generates a product with a twisted chromophore, corroborating the observed "turn-off" fluorescence response. Thus, this study extends the color palette of genetically encoded peroxynitrite biosensors, provides insight into the response mechanism of the new biosensor, and demonstrates the versatility of using protein scaffolds to modulate chemoreactivity.

Biogenesis aberration: One of the mechanisms of thrombocytopenia in COVID-19.

Background: The pathogenesis of COVID-19, including thrombocytopenia, has not been fully clarified. The lungs are a major organ of platelet production and thrombocytopenia induced by severe COVID-19 was proposed. Methods: the change of platelet level was analysed with clinical parameters in 95 hospitalized COVID-19 patients in Wuhan Third Hospital. The production of platelets in the lungs was explored in an ARDS rat model. Results: The level of platelets was negatively correlated with disease severity and was recovered with disease improvement. The non-survivors were accompanied by lower levels of platelet. The odds ratio (OR) of the valley level of the platelet count (PLTlow) was greater than 1, suggesting that PLTlow could be a death exposure factor. The platelet/lymphocyte ratio (PLR) was positively associated with severity of COVID-19, and the platelet/lymphocyte ratio threshold of 248.5 was best correlated with death risk (sensitivity 0.641 and specificity 0.815). To demonstrate the possible biogenesis aberration of platelet in lungs, an LPS-induced ARDS rat model was applied. Lower level of platelet in peripheral and less production of platelet from lungs in ARDS were demonstrated. Though megakaryocyte (MK) number in ARDS lungs is higher than controls, the immature platelet fraction (IPF) in postpulmonary blood is still at the same level as prepulmonary in ARDS rat, indicating that ARDS rats generated fewer platelets in lungs. Conclusion: Our data suggested that COVID-19-induced severe lung inflammation may impair platelet production in the lung. Thrombocytopenia may be mainly caused by platelet consumption for multiorgan thrombosis; however, biogenesis aberration of platelet in the lung induced by diffuse interstitial pulmonary damage cannot be ruled out.

Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation.

BACKGROUND: Among six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers. RESULTS: In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious mutations, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records. CONCLUSIONS: The identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos.

[Optimization of ethanol reflux extraction process of Ziziphi Spinosae Semen- Schisandrae Sphenantherae Fructus based on network pharmacology combined with response surface methodology].

The present study optimized the ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair by network pharmacology and Box-Behnken method. Network pharmacology and molecular docking were used to screen out and verify the potential active components of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus, and the process evaluation indexes were determined in light of the components of the content determination under Ziziphi Spinosae Semen and Schisandrae Sphenantherae Fructus in the Chinese Pharmacopoeia(2020 edition). The analytic hierarchy process(AHP) was used to determine the weight coefficient of each component, and the comprehensive score was calculated as the process evaluation index. The ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus was optimized by the Box-Behnken method. The core components of the Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair were screened out as spinosin, jujuboside A, jujuboside B, schisandrin, schisandrol, schisandrin A, and schisandrin B. The optimal extraction conditions obtained by using the Box-Behnken method were listed below: extraction time of 90 min, ethanol volume fraction of 85%, and two times of extraction. Through network pharmacology and molecular docking, the process evaluation indexes were determined, and the optimized process was stable, which could provide an experimental basis for the production of preparations containing Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus.

The genetic diversity, replication, and transmission of 2009 pandemic H1N1 viruses in China.

Background: The 2009 pandemic H1N1 influenza A virus (pdm09) continue to evolve, and few studies have systemically analyzed the evolution, replication, and transmission of pmd09 viruses in China. Methods: To better understand the evolution and pathogenicity of pdm09 viruses, we systematically analyzed viruses that were confirmed in 2009-2020 in China and characterized their replication and transmission ability. We extensively analyzed the evolution characteristics of pdm/09 in China over the past decades. The replication ability of 6B.1 and 6B.2 lineages on Madin-Darby canine kidney (MDCK) and human lung adenocarcinoma epithelial (A549) cells and their pathogenicity and transmission in guinea pigs were also compared. Results: In total, 3,038 pdm09 viruses belonged to clade 6B.1 (62% of all pdm09 viruses) and clade 6B.2 (4%). Clade 6B.1 pdm09 viruses are the predominant clade, with proportions of 54.1%, 78.9%, 57.2%, 58.6%, 61.7%, 76.3%, and 66.6% in the North, Northeast, East, Central, South, Southwest, and Northeast regions in China, respectively. The isolation proportion of clade 6B.1 pdm/09 viruses was 57.1%, 74.3%, 96.1%, 98.2%, 86.7%, and 78.5% in 2015-2020, respectively. A clear differentiation time point appeared in 2015 before which the evolution trend of pdm09 viruses in China was similar to that in North America but then showed a different trend after that point. To characterize pdm09 viruses in China after 2015, we further analyzed 33 pdm09 viruses isolated in Guangdong in 2016-2017, among which A/ Guangdong/33/2016 and A/Guangdong/184/2016 (184/2016) belonged to clade 6B.2, and the other 31 strains belonged to clade 6B.1. A/Guangdong/887/2017 (887/2017) and A/Guangdong/752/2017 (752/2017) (clade 6B.1), 184/2016 (clade 6B.2) and A/California/04/2009 (CA04) replicated efficiently in MDCK cells and A549 cells, as well as the turbinates of guinea pigs. 184/2016 and CA04 could transmit among guinea pigs through physical contact. Conclusion: Our findings provide novel insights into the evolution, pathogenicity, and transmission of pdm09 virus. The results show that enhancing surveillance of pdm09 viruses and timely evaluation of their virulence are essential.

All-optical image edge detection based on the two-dimensional photonic spin Hall effect in anisotropic metamaterial.

Optical image processing based on the photonic spin Hall effect (SHE) has been gaining significant attention as a convenient and an accurate way for image edge detection. However, the recent edge imaging techniques depending on optical differentiation is mainly achieved by modulation of one-dimensional photonic SHE. Here, we theoretically predict the two-dimensional photonic SHE in the anisotropic metamaterial, and find that its longitudinal and transverse displacements exhibit spin-dependent property at filling factors with increasing incidence angle. As the transverse and in-plane displacements induced by the photonic SHE can be controlled by the filling factor of the crystal structure, the optical axis angle, and the incident angle, this intrinsical effect can be used to realize a tunable edge imaging. Interestingly, by changing the optical axis of the anisotropic metamaterial, the in-plane displacements are equal to the transverse displacements for a certain filling factor and the corresponding incident angle. Therefore, we propose a two-dimensional image edge detection method based on the photonic SHE in anisotropic metamaterial. Further numerical results validate the theoretical proposal.

A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging.

cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a Kd of 2.17 µM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted ß-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

Megaproject Environmentally Responsible Behavior in China: A Test of the Theory of Planned Behavior.

Construction megaprojects play a significant role in today's infrastructure provision in terms of sustainable development, and their increasing proliferation worldwide means the environmentally responsible behavior (ERB) of those involved are becoming of vital importance. This study investigates how ERB can be best supported in megaprojects by first identifying the motivational factors that are involved based on the theory of planned behavior (TPB), followed by a survey of 188 managers involved in China megaprojects to test the TPB model using Partial Least Squares Structural Equation Modeling (PLS-SEM). The results strongly support the TPB model's predictive ability, with subjective norms being the strongest predictors, followed by attitudes and perceived behavioral control. These findings provide support for decision makers in helping to cultivate and improve the level of megaproject ERB in China and many other such countries that are similarly involved.

Detecting Key Functional Components Group and Speculating the Potential Mechanism of Xiao-Xu-Ming Decoction in Treating Stroke.

Stroke is a cerebrovascular event with cerebral blood flow interruption which is caused by occlusion or bursting of cerebral vessels. At present, the main methods in treating stroke are surgical treatment, statins, and recombinant tissue-type plasminogen activator (rt-PA). Relatively, traditional Chinese medicine (TCM) has widely been used at clinical level in China and some countries in Asia. Xiao-Xu-Ming decoction (XXMD) is a classical and widely used prescription in treating stroke in China. However, the material basis of effect and the action principle of XXMD are still not clear. To solve this issue, we designed a new system pharmacology strategy that combined targets of XXMD and the pathogenetic genes of stroke to construct a functional response space (FRS). The effective proteins from this space were determined by using a novel node importance calculation method, and then the key functional components group (KFCG) that could mediate the effective proteins was selected based on the dynamic programming strategy. The results showed that enriched pathways of effective proteins selected from FRS could cover 99.10% of enriched pathways of reference targets, which were defined by overlapping of component targets and pathogenetic genes. Targets of optimized KFCG with 56 components can be enriched into 166 pathways that covered 80.43% of 138 pathways of 1,012 pathogenetic genes. A component potential effect score (PES) calculation model was constructed to calculate the comprehensive effective score of components in the components-targets-pathways (C-T-P) network of KFCGs, and showed that ferulic acid, zingerone, and vanillic acid had the highest PESs. Prediction and docking simulations show that these components can affect stroke synergistically through genes such as MEK, NFκB, and PI3K in PI3K-Akt, cAMP, and MAPK cascade signals. Finally, ferulic acid, zingerone, and vanillic acid were tested to be protective for PC12 cells and HT22 cells in increasing cell viabilities after oxygen and glucose deprivation (OGD). Our proposed strategy could improve the accuracy on decoding KFCGs of XXMD and provide a methodologic reference for the optimization, mechanism analysis, and secondary development of the formula in TCM.

Mechanistic Insight into Diosmin-Induced Neuroprotection and Memory Improvement in Intracerebroventricular-Quinolinic Acid Rat Model: Resurrection of Mitochondrial Functions and Antioxidants.

Neurodegeneration is the final event after a cascade of pathogenic mechanisms in several brain disorders that lead to cognitive and neurological loss. Quinolinic acid (QA) is an excitotoxin derived from the tryptophan metabolism pathway and is implicated in several ailments, such as Alzheimer's, Parkinson's, Huntington's, and psychosis disease. Diosmin (DSM) is a natural flavonoid possessing such properties that may halt the course of neurodegenerative progression. In past studies, free radical scavenging, along with properties, such as antihyperglycemic, anti-inflammatory, and vasoactive properties, of DSM were pragmatic. Hence, in the current experimentations, the neuroprotective activity of DSM was investigated in the QA rat prototype. QA was administered through the intracerebroventricular route (QA-ICV) in rats on day one, and DSM (50 and 100 mg/kg, intraperitoneal route) was given from day 1 to 21. Memory, gait, sensorimotor functions, and biomarkers of oxidative mutilation and mitochondrial functions were evaluated in the whole brain. Results showed significant deterioration of sensorimotor performance, gait, and working- and long-term memory in rats by QA-ICV. These behavioral anomalies were significantly attenuated by DSM (50 and 100 mg/kg) and donepezil (standard drug). QA-ICV-induced decrease in body mass (g), diet, and water ingestion were also attenuated by DSM or donepezil treatments. QA-ICV inhibited mitochondrial complex I and II activities that caused an increase in oxidative and nitrosative stress along with a reduction in endogenous antioxidants in the brain. DSM dose-dependently ameliorated mitochondrial functions and decreased oxidative stress in QA-ICV-treated rats. DSM can be a possible alternative in treating neurodegenerative disorders with underlying mitochondrial dysfunction pathology.

Corrigendum: Detecting key functional components group and speculating the potential mechanism of Xiao-Xu-Ming decoction in treating stroke.

[This corrects the article DOI: 10.3389/fcell.2022.753425.].

Two new species of nematodes from shallow and deep-water sediments in the South China Sea.

Two new species of free-living marine nematodes from the South China Sea are described: Gomphionema setiforme sp. nov. from intertidal sediment and Paracomesoma longissimum sp. nov. from upper continental slope sediment. Gomphionema setiforme sp. nov. is characterized by the punctated cuticle without lateral differentiation and cuticular pores; short cephalic setiform sensilla; spiral amphideal fovea with 4 turns close to the anterior body extremity; large cuticularized buccal cavity with a massive dorsal tooth; pharynx with a large posterior bulb; short, curved spicules; knife-like gubernaculum without apophyses and 11 cup-shaped precloacal supplements. Paracomesoma longissimum sp. nov. is characterized by cuticle with transverse rows of fine punctuations without lateral differentiation; buccal cavity with three teeth; lateral outer labial setiform sensilla 1.5 times as long as the other four outer labial setiform sensilla; spiral amphideal fovea with 2.5 turns; spicules 350360 m long or 15.716 times cloacal body diameter; plate-like gubernaculum without apophyses and 2528 papilliform precloacal supplements.

Development, Status Quo, and Challenges to China's Health Informatization During COVID-19: Evaluation and Recommendations.

By applying advanced health information technology to the health care field, health informatization helps optimize health resource allocation, improve health care services, and realize universal health coverage. COVID-19 has tested the status quo of China's health informatization, revealing challenges to the health care system. This viewpoint evaluates the development, status quo, and practice of China's health informatization, especially during COVID-19, and makes recommendations to address the health informatization challenges. We collected, assessed, and evaluated data on the development of China's health informatization from five perspectives-health information infrastructure, information technology (IT) applications, financial and intellectual investment, health resource allocation, and standard system-and discussed the status quo of the internet plus health care service pattern during COVID-19. The main data sources included China's policy documents and national plans on health informatization, commercial and public welfare sources and websites, public reports, institutional reports, and academic papers. In particular, we extracted data from the 2019 National Health Informatization Survey released by the National Health Commission in China. We found that China developed its health information infrastructure and IT applications, made significant financial and intellectual informatization investments, and improved health resource allocations. Tested during COVID-19, China's current health informatization system, especially the internet plus health care system, has played a crucial role in monitoring and controlling the pandemic and allocating medical resources. However, an uneven distribution of health resources and insufficient financial and intellectual investment continue to challenge China's health informatization. China's rapid development of health informatization played a crucial role during COVID-19, providing a reference point for global pandemic prevention and control. To further promote health informatization, China's health informatization needs to strengthen top-level design, increase investment and training, upgrade the health infrastructure and IT applications, and improve internet plus health care services.

Corneal Recovery Following Rabbit Peripheral Blood Mononuclear Cell-Amniotic Membrane Transplantation with Antivascular Endothelial Growth Factor in Limbal Stem Cell Deficiency Rabbits.

Background: Limbal stem cell deficiency (LSCD) is a refractory ocular surface disorder characterized by progressive corneal epithelial degeneration, conjunctivalization, and neovascularization, potentially leading to blindness. There are currently no effective therapeutic options for patients experiencing routine symptomatic treatment failure. Transplantation of amniotic membrane (AM) with adherent stem cells (but not bare AM transplantation alone) has shown promise in preclinical studies for ocular surface restoration. A major limitation, however, is finding a reliable stem cell source. Stem cells can be isolated from the peripheral blood mononuclear cell (PBMC) population, and these PBMC-derived stem cells have numerous advantages over allogeneic and other autologous stem cell types for therapeutic application, including relative ease of acquisition, nonimmunogenicity, and the absence of ethical issues associated with embryonic stem cells. Experiment: We examined the efficacy of autologous PBMC-AM sheet cultures combined with postoperative antiangiogenesis treatment for corneal restoration in LSCD model rabbits. Rabbit PBMCs (rPBMCs) were isolated, labeled with EdU for in vivo tracing, and then cultured on AMs in conditioned medium before transplantation. Rabbits were transplanted with bare AMs (group 1), rPBMC-AM sheets (group 2), or rPBMC-AM sheets plus postoperative treatment with the vascular endothelial growth factor antagonist bevacizumab (group 3). Corneal opacity and neovascularization were monitored by slit-lamp imaging for 8 weeks and corneas were examined histologically at 1 and 2 months. Results: Corneal opacity decreased in all three groups over 8 weeks, but was significantly lower in group 2 and even lower in group 3. Corneal neovascularization was significantly higher in group 1 throughout the observation period, and significantly lower in group 3 than group 1 and 2 by 8 weeks post-transplant. At 4 weeks, the corneal surface completed epithelialization (although thinner than normal) in group 3 but still patchy in groups 1 and 2. By 8 weeks, the epithelium in group 3 was complete and smooth, resembling a normal epithelium. Integrin ß1 as a progenitor marker was also generally higher in groups 2 and 3. Conclusions: Autologous rPBMC-AM sheets with post-transplant topical bevacizumab can effectively facilitate corneal epithelium recovery in a LSCD model, suggesting clinical utility for LSCD-related ocular surface diseases. Impact statement Limbal stem cell deficiency (LSCD) increases corneal opacity and vascularization, resulting in severe visual impairment or even blindness. Traditional surgical limbal transplant is currently the main treatment option for LSCD, but carries the risks of rejection and immunosuppressant side effects. Autologous stem cell-based therapy is a promising alternative approach, but a reliable stem cell source is a major limitation. We report that transplantation of autologous rabbit peripheral blood mononuclear cell-amniotic membrane sheets plus antivascular endothelial growth factor restored avascular transparent cornea in a rabbit LSCD model. These results demonstrate a potentially effective approach for ocular surface reconstruction in bilateral LSCD.