Potential targets and therapeutics for cancer stem cell-based therapy against drug resistance in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common digestive malignancyin the world, which is frequently diagnosed at late stage with a poor prognosis. For most patients with advanced HCC, the therapeutic options arelimiteddue to cancer occurrence of drug resistance. Hepatic cancer stem cells (CSCs) account for a small subset of tumor cells with the ability of self-renewal and differentiationin HCC. It is widely recognized that the presence of CSCs contributes to primary and acquired drug resistance. Therefore, hepatic CSCs-targeted therapy is considered as a promising strategy to overcome drug resistance and improve therapeutic outcome in HCC. In this article, we review drug resistance in HCC and provide a summary of potential targets for CSCs-based therapy. In addition, the development of CSCs-targeted therapeuticsagainst drug resistance in HCC is summarized in both preclinical and clinical trials. The in-depth understanding of CSCs-related drug resistance in HCC will favor optimization of the current therapeutic strategies and gain encouraging therapeutic outcomes.

Approaches to Improve the Translation of Safety, Pharmacokinetics and Therapeutic Index of ADCs.

Antibody-drug conjugates (ADCs) are an important class of cancer therapies. They are complex molecules, comprising an antibody, a cytotoxic payload, and a linker. ADCs intend to confer high specificity by targeting a unique antigen expressed predominately on the surface of the tumor cells than on the normal cells and by releasing the potent cytotoxic drug inside the tumor causing cytotoxic cell death. Despite high specificity to tumor antigens, many ADCs are associated with off-target and on-target off-tumor toxicities, often leading to safety concerns before achieving the desirable clinical efficacy. Therefore, it is crucial to improve the therapeutic index (TI) of ADCs to enable the full potential of this important therapeutic modality.The review summarizes current approaches to improve the translation of safety, pharmacokinetics, and TI of ADCs. Common safety findings of ADCs resulting from off-target and on-target toxicities and nonclinical approaches to de-risk ADC safety will be discussed; multiple approaches of using preclinical and clinical dose and exposure data to calculate TI to guide clinical dosing will be elaborated; different approaches to improve TI of ADCs, including selecting the right target, right payload-linker and patients, optimizing physicochemical properties, and using fractionation dosing, will also be discussed.

Uniform design and deep learning based liquid lens optimization strategy toward improving dynamic optical performance and lowering driving force.

An efficient optimization strategy for liquid lens combining the uniform design and the deep learning is proposed to achieve improved dynamic optical performance and lowering driving force simultaneously. The membrane of the liquid lens is designed into a plano-convex cross-section, in which the contour function of the convex surface as well as the central membrane thickness is especially optimized. The uniform design method is initially utilized to select a part of uniformly distributed and representative parameter combinations from all possible parameter range, and their performance data is then obtained through simulation using MATLAB to control COMSOL and ZEMAX. After that, a deep learning framework is employed to build a four-layer neural network with its input and output layer representing the parameter combinations and the performance data, respectively. After 5 × 103 epochs, the deep neural network has undergone sufficient training, demonstrating effective performance prediction capability for all parameter combinations. Finally, a "globally" optimized design can be obtained by setting appropriate evaluation criteria which take the spherical aberration, the coma and the driving force into consideration. Compared with the conventional design using uniform membrane thickness of 100 µm and 150 µm as well as the previously reported "locally" optimized design, distinct improvements in the spherical and the coma aberrations across the entire focal length tuning range have been achieved, whilst the required driving force is largely reduced. In addition, the "globally" optimized design exhibits the best modulation transfer function (MTF) curves and provides the best image quality.

Single optical fiber based forward-viewing all-optical ultrasound self-transceiving probe.

All-optical ultrasound probes with fully integrated ultrasound generation and detection functions demonstrate some unique advantages over traditional electroacoustic counterparts. However, due to the lack of an effective solution, the most commonly used method is to assemble two separate functional optical fibers together for ultrasound generation and detection, respectively. In this Letter, an innovative strategy, to the best of our knowledge, is developed to integrate the photoacoustic effect based ultrasound generation and the Fabry-Pérot (FP) interference based ultrasound detection structures together at the end of a single double clad optical fiber (DCF), so as to make a compact forward-viewing ultrasound self-transceiving probe (1-mm diameter). From the experiment results, the as-fabricated probe can generate an ultrasound signal with an amplitude of 2.36 MPa at 2.25 mm in the transmitting mode, and its peak frequency and -6-dB bandwidth are measured to be 10.64 MHz and 22.93 MHz, respectively. When being operated under the receiving mode, the probe has a detection sensitivity of 208.4 mV/MPa for ultrasound signals with the peak frequency of 8.24 MHz, and the noise equivalent pressure (NEP) is 76.8 kPa. In addition, the forward-viewing format ultrasound self-transceiving experiment is also performed and the pulse-echo signal varying with the transmission distance is successfully captured for the first time.

AlN-based piezoelectric MEMS deformable mirror.

An AlN-based piezoelectric micro-electromechanical system (MEMS) continuous membrane deformable mirror (DM) prototype is presented for the first time. Its effective aperture diameter is 5 mm and it is equipped with 25 independently controlled actuators. Owing to the advantages associated with the AlN piezoelectric thin-film technology, attractive characteristics including CMOS compatible fabrication, bidirectional linear and negligible hysteresis actuation, and excellent linear superposition control capability have been successfully demonstrated. Moreover, good optical aberration correction performance is also validated via the surface contour fitting experiment to the Zernike polynomials up to the first 14 orders despite the non-optimized device structure design, representing great application perspective.

Characterizing the Pharmacokinetics and Biodistribution of Therapeutic Proteins: An Industry White Paper.

Characterization of the pharmacokinetics and biodistribution of therapeutic proteins (TPs) is a hot topic within the pharmaceutical industry, particularly with an ever-increasing catalog of novel modality TPs. Here, we review the current practices, and provide a summary of extensive cross-company discussions as well as a survey completed by International Consortium for Innovation and Quality members on this theme. A wide variety of in vitro, in vivo and in silico techniques are currently used to assess pharmacokinetics and biodistribution of TPs, and we discuss the relevance of these from an industry perspective, focusing on pharmacokinetic/pharmacodynamic understanding at the preclinical stage of development, and translation to human. We consider that the 'traditional in vivo biodistribution study' is becoming insufficient as a standalone tool, and thorough characterization of the interaction of the TP with its target(s), target biology, and off-target interactions at a microscopic scale are key to understand the overall biodistribution on a full-body scale. Our summary of the current challenges and our recommendations to address these issues could provide insight into the implementation of best practices in this area of drug development, and continued cross-company collaboration will be of tremendous value. SIGNIFICANCE STATEMENT: The Innovation and Quality Consortium Translational and ADME Sciences Leadership Group working group for the absorption, distribution, metabolism, and excretion of therapeutic proteins evaluates the current practices and challenges in characterizing the pharmacokinetics and biodistribution of therapeutic proteins during drug development, and proposes recommendations to address these issues. Incorporating the in vitro, in vivo and in silico approaches discussed herein may provide a pragmatic framework to increase early understanding of pharmacokinetic/pharmacodynamic relationships, and aid translational modeling for first-in-human dose predictions.

Absorption, Distribution, Metabolism, and Excretion of Therapeutic Proteins: Current Industry Practices and Future Perspectives.

Therapeutic proteins (TPs) comprise a variety of modalities, including antibody-based drugs, coagulation factors, recombinant cytokines, enzymes, growth factors, and hormones. TPs usually cannot traverse cellular barriers and exert their pharmacological activity by interacting with targets on the exterior membrane of cells or with soluble ligands in the tissue interstitial fluid/blood. Due to their large size, lack of cellular permeability, variation in metabolic fate, and distinct physicochemical characteristics, TPs are subject to different absorption, distribution, metabolism, and excretion (ADME) processes as compared with small molecules. Limited regulatory guidance makes it challenging to determine the most relevant ADME data required for regulatory submissions. The TP ADME working group was sponsored by the Translational and ADME Sciences Leadership Group within the Innovation and Quality (IQ) consortium with objectives to: (1) better understand the current practices of ADME data generated for TPs across IQ member companies, (2) learn about their regulatory strategies and interaction experiences, and (3) provide recommendations on best practices for conducting ADME studies for TPs. To understand current ADME practices and regulatory strategies, an industry-wide survey was conducted within IQ member companies. In addition, ADME data submitted to the U.S. Food and Drug Administration was also collated by reviewing regulatory submission packages of TPs approved between 2011 and 2020. This article summarizes the key learnings from the survey and an overview of ADME data presented in biologics license applications along with future perspectives and recommendations for conducting ADME studies for internal decision-making as well as regulatory submissions for TPs. SIGNIFICANCE STATEMENT: This article provides comprehensive assessment of the current practices of absorption, distribution, metabolism, and excretion (ADME) data generated for therapeutic proteins (TPs) across the Innovation and Quality participating companies and the utility of the data in discovery, development, and regulatory submissions. The TP ADME working group also recommends the best practices for condu-cting ADME studies for internal decision-making and regulatory submissions.

Overexpression of hsa_circ_0022742 suppressed hyperglycemia-induced endothelial dysfunction by targeting the miR-503-5p/FBXW7 axis.

Type I and II diabetes adversely affect the microvasculature of several organs, although the regulatory mechanisms remain unclear. Previous studies have found that differentially expressed circRNAs associated with hyperglycemia (HG) induce endothelial dysfunction. In the present study, high-throughput sequencing was employed to assess abnormal circRNA expression in human umbilical vein endothelial cells (HUVECs) after HG treatment. Then, bioinformatics analysis, luciferase reporting analysis, angiogenic differentiation analysis, flow cytometry, and qRT-PCR analysis were performed to investigate the underlying regulatory mechanism and targets. The results demonstrate that hsa_circ_0022742 expression in HUVECs was decreased by HG treatment and overexpression of hsa_circ_0022742 suppressed HG-induced endothelial dysfunction. Luciferase analysis showed that miR-503-5p and FBXW7 were downstream targets of hsa_circ_0022742. Both overexpression of FBXW7 and inhibition of miR-503-5p reversed the protective effect of hsa_circ_0022742 against HG-induced endothelial dysfunction, including apoptosis, abnormal vascular differentiation, and secretion of inflammatory factors, indicating that hsa_circ_0022742 enhanced FBXW7 expression by sponging miR-503-5p. Taken together, these findings demonstrate that overexpression of hsa_circ_0022742 suppressed HG-induced endothelial dysfunction by targeting the miR-503-5p/FBXW7 axis.

Binary amplitude switch for photoacoustic transducer toward dynamic spatial acoustic field modulation.

Photoacoustic (PA) transducers are an attractive method of producing high-amplitude, high-frequency, broad-bandwidth ultrasound signals with excellent immunity to electromagnetic interference, when compared with their traditional electroacoustic counterparts. However, the lack of effective control over the spatial sound field prohibits PA transducer technology from further widespread application. This paper presents the first, to the best of our knowledge, experimental study on the dynamic spatial ultrasound modulation strategy for the use of PA transducers, in which a novel PA transducer element is designed. This consists of a suspended compound PA conversion film, whose backing condition can be switched between air and glass through pneumatic actuation to create destructive and constructive acoustic wave interference, respectively. As a result, nearly an order of magnitude contrast in the output acoustic amplitude can be obtained by switching the device's backing condition given the same laser excitation, thus achieving a binary amplitude tuning. Furthermore, a linear PA transducer array consisting of three independently controllable elements is used for a proof-of-concept demonstration of the dynamic spatial sound field manipulation. To the best of the authors' knowledge, this is the first time that such a unique capability has been successfully applied to PA transducer technology.

Three new categories of hypoglycaemic agents and various cardiovascular diseases: A meta-analysis.

WHAT IS KNOWN AND OBJECTIVE: New hypoglycaemic agents consist of dipeptidyl peptidase four inhibitors (DPP4is), glucagon-like peptide one receptor agonists (GLP1RAs) and sodium-glucose cotransporter two inhibitors (SGLT2is). We aimed to define the association between each category of these new hypoglycaemic drugs and various cardiovascular diseases. METHODS: Large randomized trials comparing DPP4is, GLP1RAs or SGLT2is with placebo were included. Outcomes of interest were 95 kinds of cardiovascular diseases. Meta-analysis was conducted to generate pooled risk ratio (RR) and 95% confidence interval (CI). RESULTS AND DISCUSSION: Twenty-one large randomized trials were included in this meta-analysis. Compared with placebo, SGLT2is were associated with the lower risks of hypertension (RR 0.67, 95% CI 0.49-0.93), atrial fibrillation (RR 0.78, 95% CI 0.67-0.91), bradycardia (RR 0.60, 95% CI 0.40-0.89) and heart failure (RR 0.74, 95% CI 0.68-0.80); GLP1RAs were associated with the lower risk of peripheral arterial occlusive disease (RR 0.73, 95% CI 0.56-0.97) and with the higher risk of deep vein thrombosis (RR 2.12, 95% CI 1.32-3.4), while DPP4is were associated with the lower risk of peripheral ischaemia (RR 0.57, 95% CI 0.37-0.89). WHAT IS NEW AND CONCLUSIONS: Our meta-analysis revealed that SGLT2is were associated with the lower risks of hypertension, atrial fibrillation, bradycardia and heart failure; GLP1RAs were associated with the lower risk of peripheral arterial occlusive disease and with the higher risk of deep vein thrombosis, while DPP4is were associated with the lower risk of peripheral ischaemia. These findings propose that each category of these new hypoglycaemic agents should be avoided or preferred in patients at high risks of specific cardiovascular diseases.

An unexpected complication of prophylactic esophageal stenting: esophageal stent impaction after thread dislocation.

A 47-year-old woman was referred to our department with opportunistic endoscopic findings of two submucosal esophageal bulges, approximately half the circumference of the esophagus, both nearly 2.0 cm in size, and 24-27 cm from the incisors. Ultrasound endoscopy diagnosed smooth muscle tumors originating from the muscularis propria layer and she next underwent submucosal tunneling endoscopic resection. Intraoperatively, part of the tumor could not be separated from the muscularis propria layer and a U-shaped tumor was finally resected. A fully covered self-expanding esophageal nitinol stent was then inserted, covering the full circumference esophageal mucosa. The stent was fixed by ears with knotted thread and proton pump inhibitors were given for 1 week.

Bi-layered composite gratings with high diffraction efficiency enabled by near-field coupling.

In this paper, we present a design method for bi-layered composite gratings to achieve high diffraction efficiency. These composite gratings feature strong near-field coupling between their constituent dielectric subwavelength gratings, thus enabling high-efficiency first-order diffraction in the far-field. An intuitive explanation based on a wavevector matching condition for such high diffraction efficiency composite gratings is provided. According to theoretical analysis, a design strategy for the proposed composite gratings is developed and verified by numerical simulations with gratings working in both TE and TM modes. The proposed strategy could open door to develop bi-layered composite gratings for manipulating diffracted waves with high efficiency, thus may potentially enable new applications in photonic systems.

Air-backed photoacoustic transmitter for significantly improving negative acoustic pressure output.

Aiming to pursue an ultrasound signal with a significantly improved negative acoustic pressure level, which is one of the critical characteristics for exciting the ultrasound cavitation effect, a real applicable air-backed photoacoustic transmitter is presented. Different from the conventional solution of relying on a complicated focusing structure design, it works based on an acoustic signal phase reversal and amplitude superposition strategy. By using an innovative sandwich-like suspending photoacoustic layer with optimized structure design, the initial backward-propagating positive sound pressure can be converted into the forward-propagating negative one efficiently. For proof-of-concept demonstration, photoacoustic transmitter prototypes adopting a polydimethylsiloxane (PDMS)/candle soot nanoparticle/PDMS-PDMS composite as a photoacoustic conversion layer were fabricated and characterized. From experiment results, an acoustic signal with a remarkable ratio of negative pressure level to a positive one of 1.3 was successfully realized, which is the largest value ever reported, to the best of our knowledge. Moreover, when compared to the commonly used glass and PDMS-backing conditions in the photoacoustic area, nearly 200% and 400% enhancements in negative pressure output were achieved, respectively.

Ocular and systemic pharmacokinetics of BI-X, a nanobody targeting VEGF and Ang-2, after intravitreal dosing in cynomolgus monkeys - Evidence for half-life extension by albumin.

Half-life extension strategies to reduce the intravitreal dosing frequency of biomolecules for the treatment of retinal neovascular diseases are attracting increasing interest. This study investigated ocular and systemic pharmacokinetics of the trivalent nanobody BI-X (with affinity to VEGF, Ang-2 and human albumin) in cynomolgus monkeys after intravitreal injection. BI-X concentrations were measured in serial samples of plasma, vitreous humor, aqueous humor and retina. Ocular pharmacokinetics of BI-X exhibited two phases. Initially up to 2-4 weeks after dosing, BI-X concentrations in vitreal, aqueous humor and retina declined with half-lives of around 3 days, which is comparable to macromolecules with a similar molecular weight. Thereafter, only vitreal concentrations were measurable, with a terminal half-life of 13.2 days, which is considerably longer than expected based on the BI-X molecular weight or hydrodynamic radius. It is hypothesized that binding of BI-X to low levels of intraocular albumin resulted in this half-life extension. BI-X was detectable in plasma up to 10 weeks post-dosing. Plasma pharmacokinetics of BI-X exhibited a similar biphasic disposition profile to the vitreous body, with a terminal half-life of 11.8 days, thus reflecting input kinetics from the eye. In conclusion, an important half-life extension principle based on vitreal albumin binding could be confirmed in a primate model, and the data obtained can potentially be translated to humans taking into account the differing vitreal albumin concentrations.

A unique sequential C-S or N-S inductive cleavage and retro-Diels-Alder fragmentation mechanism for alkylsulfonyl piperidine- and piperazine-containing compounds.

RATIONALE: BI 605906 undergoes a collision-induced dissociation (CID) fragmentation resulting in the loss of methylsulfinic acid and butadiene to produce a corresponding imine. The fragmentation is hypothesized to occur via inductive cleavage of the C-S bond, generating a six-membered cyclic ene, followed by the retro-Diels-Alder (RDA) reaction. The aim of this study was to provide mechanistic evidence for the proposed fragmentation by investigating the CID spectra of BI 605906 and other alkylsulfonyl piperidine- and piperazine-containing compounds. METHODS: The positive electrospray ionization tandem mass spectrometric (ESI+ -MS/MS) fragmentations of BI 605906, D9 -BI 605906, GK02935, GK02942, ketoconazole, terazosin, and homopiperazine were investigated. Additionally, incubations of BI 605906 and GK02942 in human liver microsomes (HLM) preparations were conducted. Metabolite identification experiments were performed following these incubations to investigate corresponding in vitro metabolism. RESULTS: BI 605906, D9 -BI 605906, GK02935, and GK02942 demonstrated the same fragmentation pattern by generating a respective imine ion, supporting the hypothesized inductive cleavage and subsequent RDA mechanism. Ketoconazole and terazosin, which contain either an N-acetyl or tetrahydrofuranyl piperazine group, respectively, did not demonstrate this mechanism, notably because they do not have the alkylsulfonyl moiety as a good leaving group. Although homopiperazine contains an arylsulfonyl diazepane group, and the initial step produced an unsaturated diazepane ring, the subsequent RDA reaction was unable to proceed due to the absence of a six-membered cyclic ene intermediate. Additionally, we identified oxidative metabolites of BI 605906 and GK02942 in HLM incubations utilizing the proposed fragmentation pattern. CONCLUSIONS: In the mass spectrometer, compounds containing alkylsulfonyl piperidine or piperazine groups can undergo inductive cleavage, leading to a six-membered cyclic ene intermediate. This intermediate will then form a corresponding imine ion via the RDA reaction. A practical application of this work is to utilize this fragmentation for elucidating structures of metabolites arising from parent compounds containing alkylsulfonyl piperidine or piperazine moieties.

Anthropogenic Decline of African Dust: Insights From the Holocene Records and Beyond.

African dust exhibits strong variability on a range of time scales. Here we show that the interhemispheric contrast in Atlantic SST (ICAS) drives African dust variability at decadal to millennial timescales, and the strong anthropogenic increase of the ICAS in the future will decrease African dust loading to a level never seen during the Holocene. We provide a physical framework to understand the relationship between the ICAS and African dust activity: positive ICAS anomalies push the Intertropical Convergence Zone (ITCZ) northward and decrease surface wind speed over African dust source regions, which reduces dust emission and transport. It provides a unified framework for and is consistent with relationships in the literature. We find strong observational and proxy-record support for the ICAS-ITCZ-dust relationship during the past 160 and 17,000 years. Model-projected anthropogenic increase of the ICAS will reduce African dust by as much as 60%, which has broad consequences.

Biological characteristics and whole-genome analysis of the Enterococcus faecalis phage PEf771.

Enterococcus faecalis is a common pathogen causing refractory periapical periodontitis and secondary intraradicular infections. In this study, E. faecalis YN771 isolated from a re-treated root canal at a stomatology department was used as the host bacterium and was co-cultured with wastewater from the same department and patient samples to isolate a phage that lyses E. faecalis. We studied the biological and genomic characteristics of this phage. Transmission electron microscopy showed that this phage's head is icosahedral in structure, with a head diameter of around 98.4 nm, and a contractile tail of around 228.5 nm in length and a diameter of 17.3 nm. The phage was identified as a member of the Myoviridae family and named PEf771. It is sensitive to proteinase K but resistant to chloroform and Triton X-100. Its lytic cycle is 45 min, burst size is 78, optimal multiplicity of infection is 0.1, lysis spectrum is narrow, and host strain specificity is strong. Its optimal growth temperature is 37 °C, most suitable pH is 6.0, and is sensitive to ultraviolet radiation. Whole-genome sequencing of PEf771 indicated it has a genome size of 151 052 bp, with a GC content of 36.97%, and encodes 197 proteins plus 26 tRNAs. PEf771 is most closely related to E. faecalis phage EFDG1. Phage PEf771 has strong host specificity and lytic ability, so it is important to further characterize this phage and its interaction with E. faecalis.

Up-regulated lncRNA LINC00858 facilitates proliferation and migration of gastric cancer cells.

Previous evidence has shown that the long intergenic non-protein coding RNA 858 (LINC00858) is an oncogene in non-small cell lung cancers. However, the role LINC00858 plays in gastric cancer (GC) is not clear. To illustrate the role LINC00858 plays in GC, the LINC00858 expression in GC and normal tissues was firstly detected. Then, the viability, proliferation and migration of GC BGC823 and MGC803 cells were assessed following LINC00858 knockdown by si-LINC00858 transfection. The results showed that LINC00858 had a high level of expressions in GC tissues as demonstrated by both online data and qRT-PCR assay. Also, the knockdown of LINC00858 reduced the proliferation and migration of BGC823 and MGC803 cells in vitro. Taken together, our data indicate that LINC00858 plays an oncogenic role in GC cells and might act as a potential therapeutic target for GC.

Novel application of a Z-scheme photocatalyst of Ag3PO4@g-C3N4 for photocatalytic fuel cells.

A composite of Ag3PO4@g-C3N4 with the Z-scheme structure was synthesized, and used as the photoanode in a photocatalytic fuel cell (PFC). With the help of the Z-scheme design, both the degradation of tetracycline and the output of maximum power density (Pmax) were greatly enhanced in this PFC system. The degradation rate of tetracycline in the Ag3PO4@g-C3N4 PFC was 2.53 times and 3.65 times that in the PFC systems with the Ag3PO4 photoanode and the g-C3N4 photoanode, respectively. The Pmax of the Ag3PO4@g-C3N4 PFC was 6.06 µW cm-2, which was 1.46 times and 90.4 times that of the Ag3PO4 PFC (4.16 µW cm-2) and the g-C3N4 PFC (0.067 µW cm-2), respectively. The possible mechanism was proposed. The Z-scheme photoanode could not only contribute to the separation of photogenerated carriers to achieve a high photocatalytic activity, but also reserve a good redox capacity. Additionally, aeration played an important role on the PFC performance. It was demonstrated that N2 purging facilitated the electricity generation, while O2 purging promoted the pollutant degradation.

Double mutation of BRF1 and BRF2 leads to sterility in Arabidopsis thaliana.

The TFIIB-related factor (BRF) family plays vital roles in RNA polymerase (Pol) III transcription initiation. However, little is known about the role of BRF in plants. Here, we report BRF1 and BRF2 are involved in Arabidopsis reproduction. In this study, we generated BRF1 and BRF2 double mutant plants. We found that no homozygous double mutants of brf1brf2 were obtained when brf1 and brf2 were crossed, although brf1 and brf2 mutants individually developed and reproduced normally. Further experiments revealed that heterozygous brf1/ + brf2/ + produced abnormal pollen and had no seeds in some placentas of siliques. Genetic data derived from reciprocal crosses showed that BRF2 plays a dominant role in Arabidopsis reproduction. Taken together, a double mutation of BRF1 and BRF2 results in a high degree of aborted macrogametes and microgametes and complete failure in zygote generation, ultimately leading to sterility.