Traditional development of small protein scaffolds has relied on display technologies and mutation-based engineering, which limit sequence and functional diversity, thereby constraining their therapeutic and application potential. Protein design tools have significantly advanced the creation of novel protein sequences, structures, and functions. However, further improvements in design strategies are still needed to more efficiently optimize the functional performance of protein-based drugs and enhance their druggability. Here, we extended an evolution-based design protocol to create a novel minibinder, BindHer, against the human epidermal growth factor receptor 2 (HER2). It not only exhibits super stability and binding selectivity but also demonstrates remarkable properties in tissue specificity. Radiolabeling experiments with ^99mTc, ⁶⁸Ga, and ¹⁸F revealed that BindHer efficiently targets tumors in HER2-positive breast cancer mouse models, with minimal nonspecific liver absorption, outperforming scaffolds designed through traditional engineering. These findings highlight a new rational approach to automated protein design, offering significant potential for large-scale applications in therapeutic mini-protein development.

Objective To explore the correlations of lumbar bone mineral density(BMD)and metabolic syndrome(MS)in adult males.Methods Data of low dose chest CT and quantitative CT of 13 490 adult males were retrospectively analyzed,and lumbar BMD were measured to judge whether MS existed and the degree of MS,and the correlations of lumbar BMD with MS or not and the degree of MS,as well as of lumbar BMD value and the related indicators of MS were assessed.Taken lumbar BMD as the dependent variable,the age,low density lipoprotein cholesterol(LDL-C),blood uric acid(BUA),hemoglobin(Hb)and MS or not were included in multiple linear regression analysis to observe the impact of MS and related indicators on lumbar BMD.Results Among 13 490 adult males,3 900 were found with MS(MS group),while 9 590 were found without MS(non-MS group).Significant difference of lumbar BMD was detected between groups(P=0.001).Lumbar BMD values were negatively correlated with MS(rs=-0.025,P=0.004)and the degree of MS(rs=-0.038,P＜0.001),whereas positively correlated with abdominal obesity,high triglyceride and low HDL-C or not(rs=0.024,0.061,0.036,all P＜0.001)but negatively correlated with hypertension and hyperglycemia or not(rs=-0.135,-0.104,both P＜0.05).After adjustment of age,lumbar BMD of adult males was negatively correlated with MS or not as well as LDL-C(both P＜0.05),but positively correlated with BUA and Hb(both P＜0.001).Conclusion Lumbar BMD was associated with MS in adult males.

Defensive behaviors induced by innate fear or Pavlovian fear conditioning are crucial for animals to avoid threats and ensure survival. The zona incerta (ZI) has been demonstrated to play important roles in fear learning and fear memory, as well as modulating auditory-induced innate defensive behavior. However, whether the neuronal subtypes in the ZI and specific circuits can mediate the innate fear response is largely unknown. Here, we found that somatostatin (SST)-positive neurons in the rostral ZI of mice were activated by a visual innate fear stimulus. Optogenetic inhibition of SST-positive neurons in the rostral ZI resulted in reduced flight responses to an overhead looming stimulus. Optogenetic activation of SST-positive neurons in the rostral ZI induced fear-like defensive behavior including increased immobility and bradycardia. In addition, we demonstrated that manipulation of the GABAergic projections from SST-positive neurons in the rostral ZI to the downstream nucleus reuniens (Re) mediated fear-like defensive behavior. Retrograde trans-synaptic tracing also revealed looming stimulus-activated neurons in the superior colliculus (SC) that projected to the Re-projecting SST-positive neurons in the rostral ZI (SC-ZIr^SST-Re pathway). Together, our study elucidates the function of SST-positive neurons in the rostral ZI and the SC-ZIr^SST-Re tri-synaptic circuit in mediating the innate fear response.
Mice ; Animals ; Zona Incerta/metabolism* ; Neurons/metabolism* ; Fear/physiology* ; Somatostatin/metabolism*

Complex structural variants(CSVs)are genomic alterations that have more than two breakpoints and are considered as the simultaneous occurrence of simple structural variants.How-ever,detecting the compounded mutational signals of CSVs is challenging through a commonly used model-match strategy.As a result,there has been limited progress for CSV discovery com-pared with simple structural variants.Here,we systematically analyzed the multi-breakpoint con-nection feature of CSVs,and proposed Mako,utilizing a bottom-up guided model-free strategy,to detect CSVs from paired-end short-read sequencing.Specifically,we implemented a graph-based pattern growth approach,where the graph depicts potential breakpoint connections,and pattern growth enables CSV detection without pre-defined models.Comprehensive evaluations on both simulated and real datasets revealed that Mako outperformed other algorithms.Notably,validation rates of CSVs on real data based on experimental and computational validations as well as manual inspections are around 70％,where the medians of experimental and computational breakpoint shift are 13 bp and 26 bp,respectively.Moreover,the Mako CSV subgraph effectively characterized the breakpoint connections of a CSV event and uncovered a total of 15 CSV types,including two novel types of adjacent segment swap and tandem dispersed duplication.Further analysis of these CSVs also revealed the impact of sequence homology on the formation of CSVs.Mako is publicly available at https://github.com/xjtu-omics/Mako.

Microsatellite instability (MSI) is a key biomarker for cancer therapy and prognosis. Tra-ditional experimental assays are laborious and time-consuming, and next-generation sequencing-based computational methods do not work on leukemia samples, paraffin-embedded samples, or patient-derived xenografts/organoids, due to the requirement of matched normal samples. Herein, we developed MSIsensor-pro, an open-source single sample MSI scoring method for research and clinical applications. MSIsensor-pro introduces a multinomial distribution model to quantify poly-merase slippages for each tumor sample and a discriminative site selection method to enable MSI detection without matched normal samples. We demonstrate that MSIsensor-pro is an ultrafast, accurate, and robust MSI calling method. Using samples with various sequencing depths and tumor purities, MSIsensor-pro significantly outperformed the current leading methods in both accuracyand computational cost. MSIsensor-pro is available at https://github.com/xjtu-omics/msisensor-pro and free for non-commercial use, while a commercial license is provided upon request.

BACKGROUND:Co-culture withembryonic stem cels or embryonic tissues can induce differentiation of carcinoma cels into normal epithelial cels or decreasemalignancyof carcinoma cels.Acelular embryoid bodies retain the structure and important cytokines of embryonic tissues.
OBJECTIVE:To prepare acelular embryoid bodies from mouse embryonic stem cels and to investigate their effects on differentiation of mouse Lewis lung carcinoma cels at three-dimensional culturein vitro.
METHODS:Mouse embryonic stem cels(D3)were dynamicaly cultured for 7 days to produce embryoid bodiesfolowedbydecelularization with 0.1% sodium dodecyl sulfate. Mouse Lewis lung carcinoma cels were co-cultured with acelular embryoid bodiesas test group or culturedinthree-dimensionalmatrigel mediumfor 7 days as control group, respectively. Cel proliferation and expression of E-cadherin were detected by immunohistochemical staining and western blot assay, respectively. In addition, mRNA expressions ofSlug and E-cadherin were observed using RT-PCR technology.
RESULTSAND CONCLUSION:Uniform mouse embryoid bodieswere successfuly prepared, andwere completely decelularized with sodium dodecyl sulfate. After 7-day three-dimensionalmatrigelculture, in the control group,multicelular tumor spheroidswere formed,accompanied byahigherKi67positive rate;Lewis lung carcinoma cels in the test group were repopulated in the acelular embryoid bodies showing significantly lowerKi67positive rate. Compared with the control group, the absorbance ofPaxilin in the test group was significantly smaler, and the absorbance of E-cadherin was significantly higher (P< 0.05). Besides, mRNA expressions of Slug and E-cadherin were significantly decreased and increasedin the test group compared with the control group, respectively(P< 0.05). These findings indicate that the acelular embryoid bodies can promote differentiation of mouse Lewis lung carcinoma celsinthree-dimensional culturein vitro.

An electrochemical L-lactate biosensor was fabricated by combining Platinum nanoparticles (Pt-nano) with multi-walled carbon nanotubes(MWCNTs).L-lactate oxidase(LOD) was immobilized on the surface of the glassy carbon electrode (GCE) modified with MWCNTs and Pt-nano.The surface of resulting LOD/MWCNTs/Pt-nano electrode was covered by a thin layer of sol-gel to avoid the loss of LOD and to improve the anti-interference ability.The cyclic voltammetric results indicated that MWCNTs/Pt-nano catalyst displayed a higher performance than MWCNTs.Under the optimized conditions, i.e., applied potential of 0.5 V, pH 6.4, 25 ℃, the proposed biosensor's determination range was 0.2-2.0 mmol/L, response time was within 5 s, and the sensitivity was 6.36 (A/(mmol/L).It still kept 90% activity after 4 weeks.The fabricated biosensor had practically good selectivity against interferences.The results for whole blood samples analyzed by the present biosensor showed a good agreement with those analyzed by spectrophotometric method.