SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield.

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.

Females with diabetes have a higher risk of ischemic stroke readmission: a retrospective cohort study.

BACKGROUND: There are significant sex differences in the incidence of stroke or diabetes mellitus. However, little is known about sex differences in stroke rehospitalization among diabetic patients. OBJECT: To explore the sex differences in short-term and long-term rehospitalization of ischemic stroke patients with Type 2 diabetes mellitus. METHODS: A retrospective cohort study was conducted from 2017 to 2021. The rehospitalization events of ischemic stroke patients with diabetes mellitus were identified by the national unified Electronic Health Record. Propensity score matching was applied to adjust for multiple covariates, and LASSO regression was used to screen for independent variables. Cox proportional hazards model was utilized to analyze the different sex in short-term (90 days, 1 year) and long-term (5 years) rehospitalization in ischemic stroke patients with type 2 diabetes mellitus. RESULT: A total of 10,724 ischemic stroke patients were included in this study, of whom 5,952 (55.5%) were males. After a 1:1 propensity score matching, there were 3,460 males and 2,772 females. After adjusting for confounding factors, female patients with type 2 diabetes had an increased risk of ischemic stroke rehospitalization at 90 days (HR: 1.94, 95%CI: 1.13-3.33, P < 0.05), 1 year (HR: 1.65, 95%CI:1.22-2.23, P = 0.001), and 5 years (HR: 1.58, 95%CI: 1.26-1.97, P < 0.001). However, there was no significant relationship between male patients with type 2 diabetes and the risk of ischemic stroke rehospitalization, either in the short or long term. CONCLUSION: Females with type 2 diabetes mellitus have a higher risk of ischemic stroke rehospitalization in both the short-term and long-term.

Construction of Core-Shell MOF CSMnP with Enzyme-Like Activity for Chemotherapy and Chemodynamic Therapy.

Materials with enzyme-like activity have received a lot of attention in the field of tumor catalytic therapy. Here, biocompatible core-shell MOF CSMnP with two valence states of Mn ion, which could process chemodynamic therapy (CDT), was designed and synthesized. Besides, it could also promote a series of catalytic processes in the tumor microenvironment (TME). CSMnP catalyzed endogenous hydrogen peroxide (H2O2) to oxygen (O2) via catalase-like activity and then combined with the outer layer Mn(II)-PBC to convert O2 into superoxide radicals (•O2-), exhibiting oxidase-like activity. Besides, intracellular glutathione (GSH) could be effectively consumed through the glutathione oxidase-like activity of Mn3+. The occurrence of the cascade reactions effectively amplified the enzymatic production to enhance CDT. Furthermore, the therapeutic effect of CSMnP was improved through the loading of cationic drug DOX. The loading capacity was 11.10 wt %, which was 2.2 times that of Mn(III)-PBC (4.95 wt %), and the release of DOX showed a characteristic response. Therefore, the core-shell MOF with enzyme-like activity had a potential application for tumor combination therapy.

A Cu-Based Metal-Organic Framework Cu-Cip with Cuproptosis for Cancer Therapy and Inhibition of Cancer Cell Migration.

Microflora within cancer cells plays a pivotal role in promoting metastasis of cancer. However, contemporary anticancer research often overlooks the potential benefits of combining anticancer and antibacterial agents. Consequently, a metal-organic framework Cu-Cip with cuproptosis and antibacterial properties was synthesized for cancer therapy. To enhance the anticancer effect of the material, Mn2+ was loaded into Cu-Cip, yielding Mn@Cu-Cip. The fabricated material was characterized using single-crystal X-ray diffraction, PXRD, and FT-IR. By interacting with overexpressed H2O2 to produce ROS and accumulating Cu ions in cancer cells, MOFs exhibited excellent anticancer performance. Moreover, the material displayed the function of damaging Staphylococcus aureus and Escherichia coli, revealing the admirable antibacterial properties of the material. In addition, the antibacterial ability could inhibit tumor cell migration. The Cu-based MOF revealed promising applications in the field of tumor treatment.

Design and implementation of a dual aspheric integrated shaping element for a high-power fiber laser.

A dual aspheric integrated beam shaper suitable for a high-power laser situation has been designed and realized. The model for this lens was derived theoretically and the performance was evaluated using a detailed simulation. The ultrasonic vibration assisted cutting and the high-precision grinding and polishing technology were used for the processing. The surface accuracy was less than 200 nm measured with a profiler, and the roughness was smaller than 20 nm with the help of the white light interferometer. Shaping experiments were carried out, which verified that the Gaussian beam has uniform intensity distribution with a uniformity of 85.13% in the near field and converges to a point in the far field, which is exactly as expected. It thus provides an actual selection for high-power laser shaping.

Dementia increases the risk of death in stroke patients: A retrospective cohort-based risk score model study.

BACKGROUND: The relationship between dementia and the mortality of stroke is a significant concern for patients and careers. However, there are few research about it in China and a lack of reliable data on the risk of dementia. We aim to analyze and compare the risk of death in stroke patients with and without dementia. Further investigation into the predictive value of dementia for stroke death. METHODS: All patients with stroke who were identified among residents of Ningxia, between January 1, 2014 to December 31, 2021, set death or May 22, 2022 as the observation endpoint. All patients were screened by 1:4 propensity score matching (PSM). The association between dementia and all-cause mortality was evaluated using Cox regression with survival time. Evaluation of the predictive value of dementia using decision curve analysis (DCA) and clinical impact curve (CIC) curves. RESULT: Mortality of stroke with dementia is 45.4% and without dementia is 13.8%, further calculated one-year mortality is higher in the patients with dementia than without dementia (17.3%vs. 5.4%, p < 0.001). Stroke patients with dementia had a 3.74 times higher risk of death (95% CI = 3.29,4.26) and had a shorter survival time than those without dementia. Dementia was an independent predictor of death in all models (hazard ratio [HR]=3.77,95%CI: 3.31-4.30, p < 0.001). DCA and CIC curves indicated that dementia has a high value in predicting the risk of death in stroke patients. CONCLUSION: Dementia is an independent risk factor for death and reduces survival time in stroke patients.

Erythrocyte Membrane-Coated Invisible Acoustic-Sensitive Nanoparticle for Inducing Tumor Thrombotic Infarction by Precisely Damaging Tumor Vascular Endothelium.

Selective induction of tumor thrombus infarction is a promising antitumor strategy. Non-persistent embolism due to non-compacted thrombus and activated fibrinolytic system within the tumor large blood vessels and tumor margin recurrence are the main therapeutic bottlenecks. Herein, an erythrocyte membrane-coated invisible acoustic-sensitive nanoparticle (TXA+DOX/PFH/RBCM@cRGD) is described, which can induce tumor thrombus infarction by precisely damaging tumor vascular endothelium. It is revealed that TXA+DOX/PFH/RBCM@cRGD can effectively accumulate on the endothelial surface of tumor vessels with the help of the red blood cell membrane (RBCM) stealth coating and RGD cyclic peptide (cRGD), which can be delivered in a targeted manner as nanoparticle missiles. As a kind of phase-change material, perfluorohexane (PFH) nanodroplets possess excellent acoustic responsiveness. Acoustic-sensitive missiles can undergo an acoustic phase transition and intense cavitation with response to low-intensity focused ultrasound (LIFU), damaging the tumor vascular endothelium, rapidly initiating the coagulation cascade, and forming thromboembolism in the tumor vessels. The drugs loaded in the inner water phase are released explosively. Tranexamic acid (TXA) inhibits the fibrinolytic system, and doxorubicin (DOX) eliminates the margin survival. In summary, a stealthy and acoustically responsive multifunctional nanoparticle delivery platform is successfully developed for inducing thrombus infarction by precisely damaging tumor vascular endothelium.

Screening of six cation exchange resins for high binding capacity, monomer purity and step yield: A case study.

Cation exchange (CEX) chromatography has been widely used as a polishing step in downstream processing of therapeutic monoclonal antibodies (mAbs). It has been well documented that the performance of a particular CEX resin heavily relies on buffer type, pH and conductivity. In this work, with a case study, we screened six commercial CEX resins under different pHs and conductivities. Initial screening was conducted on Tecan to find conditions under which high binding capacity was achieved. Next, performance of each resin was further evaluated using a small column under the identified optimum binding conditions. Variations in binding capacity and purity of eluate were observed among these resins. The adopted approach allows for identification of resins exhibiting good binding capacity, aggregate clearance and recovery.

Scanning measurement of surface error and thickness variation of the hemispherical resonator.

Hemispherical resonant gyroscopes (HRGs) are solid-state vibration gyroscopes with the highest precision and are widely used in the aerospace field. The core part of the gyroscope is the resonator, which is a thin-walled hemispherical shell. Surface error and thickness variation of a hemispherical shell causes frequency splitting, which degrades the performance of the HRG. In order to guide the mass leveling of hemispherical resonator, this paper presents a new method for scanning measurement of the surface error and thickness variation of hemispherical resonators. First, a multi-axis platform is designed for noncontact sensor scanning measurements along the meridian and latitudinal lines of the hemispherical resonator. Second, the error model of the measurement system is established. The surface error of the standard sphere is measured to calibrate and compensate for the assembly errors of the measuring device. In addition, the identification accuracy of assembly errors and the influence of assembly errors on thickness measurement are simulated by a computer. Finally, the surface error and thickness variation of the hemispherical resonators are measured. The method is experimentally demonstrated and validated with a wavefront interferometry test. The results show that the method can achieve high precision and high repeatability, which is instructive for assessment of the machining error and further evaluation of the hemispherical resonator.

The transcription factor NKX2-2 regulates oligodendrocyte differentiation through domain-specific interactions with transcriptional corepressors.

The homeodomain protein NK2 homeobox 2 (NKX2-2) is a transcription factor that plays a critical role in the control of cell fate specification and differentiation in many tissues. In the developing central nervous system, this developmentally important transcription factor functions as a transcriptional repressor that governs oligodendrocyte (OL) differentiation and myelin gene expression, but the roles of various NKX2-2 structural domains in this process are unclear. In this study, using in situ hybridization, immunofluorescence, and coimmunoprecipitation, we determined the structural domains that mediate the repressive functions of murine NKX2-2 and identified the transcriptional corepressors that interact with it in OL cells. Through in ovo electroporation in embryonic chicken spinal cords, we demonstrate that the N-terminal Tinman domain and C-terminal domain synergistically promote OL differentiation by recruiting distinct transcriptional corepressors, including enhancer of split Groucho 3 (GRG3), histone deacetylase 1 (HDAC1), and DNA methyltransferase 3 α (DNMT3A). We also observed that the NK2-specific domain suppresses the function of the C-terminal domain in OL differentiation. These findings delineate the distinct NKX2-2 domains and their roles in OL differentiation and suggest that NKX2-2 regulates differentiation by repressing gene expression via multiple cofactors and molecular mechanisms.

Sarcopenia is associated with the presence of nonalcoholic fatty liver disease in Zhejiang Province, China: a cross-sectional observational study.

BACKGROUND: Currently, both non-alcoholic fatty liver disease (NAFLD) and sarcopenia have attracted extensive attention in public health. However, the relationship between NAFLD and sarcopenia remains unclear. This study aimed to clarify the sex-specific association between sarcopenia and NAFLD according to the Asian Working Group for Sarcopenia (AWGS). METHODS: Dual-energy X-ray absorptiometry (DXA) and hepatic ultrasonography were measured in 578 participants (92 men and 486 women) during their annual health examinations. Multivariate logistic regression models were used to explore the association between NAFLD and sarcopenia with its two components. RESULTS: A total of 154 participants (30 men and 124 women) had NAFLD. The prevalence of sarcopenia was higher among the participants with NAFLD than among those without NAFLD (men: 20.0% vs. 9.7%, P = 0.295, women: 15.3% vs. 8.0%, P = 0.019). Low muscle mass (LMM) was independently associated with NAFLD in both men and women (men: odds ratio [OR], 2.88; 95% confidence interval [CI] 1.52-5.46; women: OR, 2.08; 95% CI 1.63-2.67). However, low muscle strength (LMS) was independently associated with NAFLD only in male participants, with an OR of 1.15 (95% CI 1.02-1.28). CONCLUSION: The occurrence of sarcopenia was associated with a higher risk of NAFLD, especially in men, as demonstrated by lower muscle mass and lower muscle strength.

[Mechanism of Huangqi Guizhi Wuwu Decoction in treatment of rheumatoid arthritis based on UPLC-LTQ-Orbitrap-MS, network pharmacology, and cell experiment].

In this study, ultra-high performance liquid chromatography-linear ion trap/electrostatic field orbit trap combined-type mass spectrometry(UPLC-LTQ-Orbitrap-MS) was used to analyze the main active components of Huangqi Guizhi Wuwu Decoction(HQGZ). A total of 50 active components were identified from HQGZ and 108 potential targets of the components related to the treatment of rheumatoid arthritis were retrieved based on network pharmacology, including 87 key targets, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. The result indicated that HQGZ may exert therapeutic effects mainly through the sphingolipid signaling pathway, tumor necrosis factor(TNF) signaling pathway, as well as the positive regulation of ribonucleic acid(RNA) polymerase Ⅱ promoter transcription, inflammatory response and other biological processes. At the same time, cell experiment was performed to verify the key proteins in the TNF signaling pathway. The results demonstrated that HQGZ significantly reduced the expression of caspase-3(CASP3), TNF, relaxed(RELA) protein, and IkappaB kinase beta(IKBKB) in fibroblast-like synoviocytes induced by TNF-α. The results of UPLC-LTQ-Orbitrap-MS, network pharmacology and cell experiment showed that the active components in HQGZ may inhibit inflammatory response and regulate immune function and cell apoptosis by modulating key proteins in TNF signaling pathway to treat rheumatoid arthritis.

Divinylsulfonamides enable the construction of homogeneous antibody-drug conjugates.

Methods that site-specifically attach payloads to an antibody with controlled DAR (Drug-Antibody Ratio) are highly desirable for the generation of homogeneous antibody-drug conjugates (ADCs). We describe the use of N-phenyl-divinylsulfonamide scaffold as a linker platform to site-specifically construct homogeneous DAR four ADCs through a disulfide re-bridging approach. Several monomethyl auristatin E (MMAE)-linkers were synthesized and the drug-linkers that contain electron-donating groups on the phenyl of the linker showed high stability. Her2-targeted MMAE-linker-herceptin and EGFR targeted MMAE-linker-cetuximab conjugates were prepared. The conjugates demonstrated high efficacy and selectivity for killing target-positive cancer cells in vitro. The EGFR-targeted conjugates also showed significant antitumor activities in vivo.

Influence of acupuncture on the expression of VIP, SP, NKA and NKB, cAMP/cGMP and HE content and treatment of bronchial asthma in rats.

This research was set up to explore the neural mechanisms of acupuncture in the treatment of bronchial asthma in rats by detecting the content of substance P(SP), vasoactive intestinal peptide (VIP), neurokinin A(NKA), neurokinin B (NKB), cyclic adenosine monophosphate/cyclic guanosine monophosphate ratio (cAMP/cGMP) and hematoxylin-eosin (HE) staining for the pathological changes of lung tissue, in order to Institute Certain Experimental and Theoretical Foundation for Traditional Chinese Medicine (TCM) Prevention and Treatment of Bronchial Asthma. For this purpose, fifty healthy adult Wistar male rats, weighing 200-250 g, were randomly divided into 5 groups: normal control group A, asthma control group B, asthma acupuncture group C, adrenalectomy (ADX)-asthma  group D, adrenalectomy (ADX)-asthma acupuncture group E. Group A was raised with other groups at the same period; Group B was induced asthma by ovalbumin; Group C was induced asthma as Group B and then acupunctured five acupoints (bilateral Feishu, bilateral Fengmen, and Dazhui); Group D was induced asthma after adrenalectomy; group E was treated with acupuncture on the basis of group D. HE staining was performed in the lung tissue of rats from each group, and histopathologic changes were observed. SP, VIP, NKA, NKB in each rat lung tissue were measured by immunohistochemistry. cAMP/cGMP was measured with ELISA to speculate the neural mechanisms of acupuncture in the treatment of bronchial asthma. The results were as: decrease of cAMP/cGMP and VIP and increase of SP, NKA, NKB in the lung tissue are the neural mechanisms of an asthma attack. The increase of cAMP/cGMP and decrease of NKA, NKB, SP and VIP in the lung tissue of group C indicated the improvement of bronchial asthma symptoms. It is possible that the decrease of NKA and NKB, increase of cAMP/cGMP and a slight change of SP and VIP in group E were related to the reduction of glucocorticoid after ADX which influenced the effect of acupuncture. The neural regulation mechanisms of acupuncture in the treatment of bronchial asthma were related to bronchiectasis caused by stimulation of adrenergic nerve and inhibition of the vagus nerve function by acupuncture, and related to the release of inflammatory mediators.

CT diagnosis of pleural and stromal invasion in malignant subpleural pure ground-glass nodules: an exploratory study.

OBJECTIVES: To assess the risk of visceral pleural invasion (VPI) and improve the diagnosis of invasive adenocarcinoma (IA) in pure ground-glass nodules (pGGNs) in contact with pleura, through a comprehensive analysis of the thin-section CT features of subpleural malignant pGGNs. METHODS: CT findings and clinical information of 115 consecutive patients in our hospital between January 2012 and December 2015 who met the following criteria were retrospectively studied: (a) thin-section CT within 1 month before surgery proved pGGN in contact with pleura, and (b) the pGGN was confirmed as malignancy by surgery. Univariate analysis and a multivariate logistic regression analysis were conducted to identify the independent risk factors of IA and VPI. RESULTS: No pleural invasion was observed microscopically in any of the pGGNs. Univariate analysis indicated that tumour shape (p = 0.004), relative density (p = 0.038) and the existence of pleural retraction (p < 0.001) were significantly different between the invasive group and pre- or minimally invasive group. Multivariate logistic regression analysis revealed that pleural retraction (OR, 5.663; p < 0.001), lobulated tumour shape (OR, 4.812; p = 0.016) and tumour relative density greater than 1.60 (OR, 4.449; p = 0.001) were independent risk factors of IA. CONCLUSIONS: Pulmonary adenocarcinoma manifesting as pGGN generally does not invade the pleura. A comprehensive consideration of tumour shape, relative density and tumour-pleural relationship can independently predict IA. KEY POINTS: • This study showed that pGGN-like adenocarcinoma generally does not invade the pleura. • This study suggested that persistent pGGN with pleural retraction, lobulated shape and high relative density (> 1.60) may very likely be invasive adenocarcinoma. • Using "relative density" can reduce confounding of contrast agent and respiratory status in analysis of CT images.

Development of a fluorescence resonance energy transfer-based intracellular assay to identify novel enterovirus 71 antivirals.

Enterovirus 71 (EV71) is considered one of the most virulent pathogens in the family Picornaviridae. However, there have been no effective treatments for the severe complications caused by EV71. Development of new drugs against targets that are essential for viral replication often requires screening large collections of compounds, for which a high-throughput screening platform is needed. In this study, a drug-screening platform was developed based on a genetically engineered cell line that displays fluorescence resonance energy transfer (FRET) and shows a real-time and quantifiable impairment of FRET upon EV71 infection. A library of small molecules consisting of 1280 compounds with defined bioactivities was used for screening drugs with anti-EV71 activity; accurate, rapid, and robust results were obtained from this screening procedure. Ten drugs were identified in the primary screening, and their antiviral activities were indicated by dose-dependent elevation of FRET. Among these, AC-93253, mitoxantrone and N-bromoacetamide had not been reported as enterovirus inhibitors, and it was confirmed that they were able to suppress viral yields in a dose-dependent manner. Taken together, these studies demonstrate the feasibility of this FRET-based platform for efficient screening and identification of novel compounds with activity against EV71 infection.

Idarubicin is a broad-spectrum enterovirus replication inhibitor that selectively targets the virus internal ribosomal entry site.

Enterovirus 71 (EV71) causes life-threatening diseases with neurological manifestations in young children. However, the treatment of EV71 infections remains an unmet medical need. Idarubicin (IDR) is an anthracycline compound that is used therapeutically for certain types of tumour. In this study, we identified IDR as an EV71 inhibitor, which displayed antiviral potency in the submicromolar range and substantially protected cells from the cytopathic effects and cell death caused by EV71 infections. The antiviral effects extended to several other enterovirus (EV) species, and these effects were independent of cytotoxicity or topoisomerase inhibition. Structure-activity relationship studies indicated the importance of the anthracycline scaffold for anti-EV potency. IDR effectively blocked the synthesis of viral protein and RNA, but not the viral proteolysis processes. Moreover, anthracyclines were demonstrated to suppress EV internal ribosomal entry site (IRES)-mediated translation; conversely, the cellular p53 IRES activity was not sensitive to IDR action. Inhibition of IRES-mediated translation by IDR correlated with the affinity of binding between IDR and the particular IRES. Moreover, IDR impaired binding between the EV71 IRES RNA and hnRNP A1, a known host IRES trans-acting factor. In sum, we have identified a USA FDA-approved anticancer drug with the new indication as a selective EV IRES binder and inhibitor. The finding may also provide leads for the development of novel antiviral therapies directed at the EV IRES RNA.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development.

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development.

[Research status on regulation of Chinese herbal compound on intestinal microecology].

The ralationship between traditional Chinese medicine (TCM) and intestinal microecology is increasingly being given more and more attention. Combined with the devolopment of intestinal microecology disciplines, effects of TCM on regulation of intestinal microecology have been gradually explained. Both clinical studies and animal experiments have confirmed that TCM can maintain the balance of intestinal microecology and regulate the intestinal flora. The author arrangemented the documents related to Chinese herbal compound adjusting intestinal flora in the recent ten years, summarized that the Chinese herbal compound which can strength spleen and replenish Qi, relax bowels and regulate Qi, dissipate dampness and check diarrhea, clear away heat and toxic materials, promote digestion and relieve stasis had certain regulation effects on intestinal microecology, providing basis for revealing the TCM essence of intestinal microecology.

Sterols are required for cell-fate commitment and maintenance of the stomatal lineage in Arabidopsis.

Asymmetric cell division is important for regulating cell proliferation and fate determination during stomatal development in plants. Although genes that control asymmetric division and cell differentiation in stomatal development have been reported, regulators controlling the process from asymmetric division to cell differentiation remain poorly understood. Here, we report a weak allele (fk-J3158) of the Arabidopsis sterol C-14 reductase gene FACKEL (FK) that shows clusters of small cells and stomata in leaf epidermis, a common phenomenon that is often seen in mutants defective in stomatal asymmetric division. Interestingly, the physical asymmetry of these divisions appeared to be intact in fk mutants, but the cell-fate asymmetry was greatly disturbed, suggesting that the FK pathway links these two crucial events in the process of asymmetric division. Sterol profile analysis revealed that the fk-J3158 mutation blocked downstream sterol production. Further investigation indicated that cyclopropylsterol isomerase1 (cpi1), sterol 14α-demethylase (cyp51A2) and hydra1 (hyd1) mutants, corresponding to enzymes in the same branch of the sterol biosynthetic pathway, displayed defective stomatal development phenotypes, similar to those observed for fk. Fenpropimorph, an inhibitor of the FK sterol C-14 reductase in Arabidopsis, also caused these abnormal small-cell and stomata phenotypes in wild-type leaves. Genetic experiments demonstrated that sterol biosynthesis is required for correct stomatal patterning, probably through an additional signaling pathway that has yet to be defined. Detailed analyses of time-lapse cell division patterns, stomatal precursor cell division markers and DNA ploidy suggest that sterols are required to properly restrict cell proliferation, asymmetric fate specification, cell-fate commitment and maintenance in the stomatal lineage cells. These events occur after physical asymmetric division of stomatal precursor cells.