The mechanism of sitagliptin inhibition of colorectal cancer cell lines' metastatic functionalities.

The cell membrane glycoprotein CD26 with peptidase activity (DPP4) and/or its soluble CD26/DPP4 counterpart expression and/or activity are altered in several cancers. Its role in metastasis development was recently highlighted by the discovery of CD26+ cancer stem cell subsets and the fact that clinical DPP4 inhibitors showed antimetastatic effects in animal models. Also, diabetic patients treated with the DPP4 inhibitor sitagliptin showed greater overall survival after colorectal or lung cancer surgery than patients under other diabetic therapies. However, the mechanism of action of these inhibitors in this context is unclear. We studied the role of CD26 and its DPP4 enzymatic activity in malignant cell features such as cell-to-cell homotypic aggregation, cancer cell motility, and invasion in a panel of human colorectal cancer (CRC) cell lines, avoiding models that include the physiological role of DPP4 in chemotaxis. Present results indicate that CD26 participates in the induction of cell invasion, motility, and aggregation of CD26-positive CRC cell lines. Moreover, only invasion and motility assays, which are collagen matrix-dependent, showed a decrease upon treatment with the DPP4 inhibitor sitagliptin. Sitagliptin showed opposite effects to those of transforming growth factor-ß1 on epithelial-to-mesenchymal transition and cell cycle, but this result does not explain its CD26/DPP4-dependent effect. These results contribute to the elucidation of the molecular mechanisms behind sitagliptin inhibition of metastatic traits. At the same time, this role of sitagliptin may help to define areas of medicine where DPP4 inhibitors might be introduced. However, they also suggest that additional tools against CD26 as a target might be used or developed for metastasis prevention in addition to gliptins.

Deploying unsupervised clustering analysis to derive clinical phenotypes and risk factors associated with mortality risk in 2022 critically ill patients with COVID-19 in Spain.

BACKGROUND: The identification of factors associated with Intensive Care Unit (ICU) mortality and derived clinical phenotypes in COVID-19 patients could help for a more tailored approach to clinical decision-making that improves prognostic outcomes. METHODS: Prospective, multicenter, observational study of critically ill patients with confirmed COVID-19 disease and acute respiratory failure admitted from 63 ICUs in Spain. The objective was to utilize an unsupervised clustering analysis to derive clinical COVID-19 phenotypes and to analyze patient's factors associated with mortality risk. Patient features including demographics and clinical data at ICU admission were analyzed. Generalized linear models were used to determine ICU morality risk factors. The prognostic models were validated and their performance was measured using accuracy test, sensitivity, specificity and ROC curves. RESULTS: The database included a total of 2022 patients (mean age 64 [IQR 5-71] years, 1423 (70.4%) male, median APACHE II score (13 [IQR 10-17]) and SOFA score (5 [IQR 3-7]) points. The ICU mortality rate was 32.6%. Of the 3 derived phenotypes, the A (mild) phenotype (537; 26.7%) included older age (< 65 years), fewer abnormal laboratory values and less development of complications, B (moderate) phenotype (623, 30.8%) had similar characteristics of A phenotype but were more likely to present shock. The C (severe) phenotype was the most common (857; 42.5%) and was characterized by the interplay of older age (> 65 years), high severity of illness and a higher likelihood of development shock. Crude ICU mortality was 20.3%, 25% and 45.4% for A, B and C phenotype respectively. The ICU mortality risk factors and model performance differed between whole population and phenotype classifications. CONCLUSION: The presented machine learning model identified three clinical phenotypes that significantly correlated with host-response patterns and ICU mortality. Different risk factors across the whole population and clinical phenotypes were observed which may limit the application of a "one-size-fits-all" model in practice.

Relevance of matrix metalloproteases in non-small cell lung cancer diagnosis.

BACKGROUND: The need for novel biomarkers that could aid in non-small cell lung cancer (NSCLC) detection, together with the relevance of Matrix Metalloproteases (MMPs) -1, -2, -7, -9 and -10 in lung tumorigenesis, prompted us to assess the diagnostic usefulness of these MMPs and the Tissue Inhibitor of Metalloproteinase (TIMP) -1 in NSCLC patients. METHODS: Markers were evaluated in an initial study cohort (19 NSCLC cases and 19 healthy controls). Those that better performed were analyzed in a larger sample including patients with benign lung diseases. Serum MMPs and TIMP-1 were determined by multiplexed immunoassays. Logistic regression was employed for multivariate analysis of biomarker combinations. RESULTS: MMPs and TIMP-1 were elevated in the serum of NSCLC patients compared to healthy controls. MMP-1, -7 and -9 performed at best and were further evaluated in the sample including benign pathologies, corroborating the superiority of MMP-9 in NSCLC discrimination, also at early-stage NSCLC. The optimal diagnostic value was obtained with the model including MMP-9, gender, age and smoking history, that demonstrated an AUC of 0.787, 85.54% sensitivity and 64.89% specificity. CONCLUSION: Our results suggest that MMP-9 is a potential biomarker for NSCLC diagnosis and its combined measurement with other biomarkers could improve NSCLC detection.

The photo-elicitation of food worlds: A study on the eating behaviors of low socioeconomic Chilean women.

Traditional methods for studying eating behaviors include quantitative methods such as 24-h dietary recalls or food frequency questionnaires. Recently, visual methods such as photo-elicitation (PE) have been recognized as useful for studying and understanding eating behaviors. PE has been defined as the use of images during an interview. The goals of this study are to demonstrate the potential of PE for exploring the eating behaviors of Chilean women of low socioeconomic status and to show the advantages and disadvantages of PE from the participants' points of view. The study included 31 participants who were asked to take pictures that represented what they considered important to them in their "food world". The pictures were developed and participants were invited to participate in an individual interview. Participants were able to talk about their eating behaviors and those of their families, the factors influencing those behaviors, their dietary knowledge and skills, and their reflections on their diet using the photographs. PE proved to be a feasible research technique for the studied population, and was well received and enjoyed by the participants. The participants perceived a few barriers with PE, such as forgetting to take pictures or not having ideas for new pictures. Nevertheless, PE allowed researchers to obtain rich information about eating behaviors, and can therefore be a useful method for working with populations of underserved areas. The PE data that this study collected could be used to create or improve interventions promoting healthy eating within the studied population.

Food and family care during the COVID-19 pandemic: A study of women's domestic workload during the first wave in Chile.

This study aimed to explore women's perceptions of domestic work related to food and family care during the first wave of the COVID-19 pandemic in Chile and its association with sociodemographic and health variables. We conducted a cross-sectional, analytical, non-probabilistic study. A sample of 2047 women answered an online self-report survey that included a Likert scale about the perception of domestic work associated with food. The survey also included an open comment section. The survey was available between May and June 2020, during the first wave of the COVID-19 pandemic and when most of the country had some degree of mobility restriction. 70.2% of participants perceived their domestic work as "regular"; being younger, having a higher educational level, caring for children or the elderly, and having worse self-perception of mental and general health status increased the chances of having a lower perception of the burden of these tasks. In comments, women declared how heavy the domestic work was, the challenges of being together with their families and of paid job requirements, and how family demands from them increased. Most women felt that their domestic work was heavier during this pandemic period: some groups of women could be at risk of being more affected by this extra workload at home. The importance of interventions and public policies with a gender perspective becomes relevant, considering the role of women in the home and the necessity to generate a social change regarding the domestic burden associated with gender.

Bacterial surface display of human lectins in Escherichia coli.

Lectin-glycan interactions sustain fundamental biological processes involved in development and disease. Owing to their unique sugar-binding properties, lectins have great potential in glycobiology and biomedicine. However, their relatively low affinities and broad specificities pose a significant challenge when used as analytical reagents. New approaches for expression and engineering of lectins are in demand to overcome current limitations. Herein, we report the application of bacterial display for the expression of human galectin-3 and mannose-binding lectin in Escherichia coli. The analysis of the cell surface expression and binding activity of the surface-displayed lectins, including point and deletion mutants, in combination with molecular dynamics simulation, demonstrate the robustness and suitability of this approach. Furthermore, the display of functional mannose-binding lectin in the bacterial surface proved the feasibility of this method for disulfide bond-containing lectins. This work establishes for the first time bacterial display as an efficient means for the expression and engineering of human lectins, thereby increasing the available toolbox for glycobiology research.

Coxiella burnetii seroprevalence in sheep herd from Paraguay: First evidence of bacterial circulation in the country.

Coxiella burnetii is the agent of Q fever, a disease that poses risks to public health and damages livestock. We discovered the circulation of C. burnetii for the first time in Paraguay, based on the seropositivity of a flock of >300 sheep. The animals were tested by IFA for anti-C. burnetii antibodies and by SAM for anti-Leptospira spp. antibodies, an important differential diagnosis for reproductive disorders in sheep in Paraguay. C. burnetii seropositivity was determined in 45%, in contrast to Leptospira spp. which had no reactive samples. Cases of miscarriage and fetal resorption were associated with high seropositivity titers. This study suggests the circulation of a unique genotype in the country and an imminent risk to public health, since in addition to being highly transmissible and infectious to humans, Q fever is still not a cause for concern on the part of government and health agencies in the country.

Au@Ag Core-Shell Nanoparticles for Colorimetric and Surface-Enhanced Raman-Scattering-Based Multiplex Competitive Lateral Flow Immunoassay for the Simultaneous Detection of Histamine and Parvalbumin in Fish.

Foodborne allergies and illnesses represent a major global health concern. In particular, fish can trigger life-threatening food allergic reactions and poisoning effects, mainly caused by the ingestion of parvalbumin toxin. Additionally, preformed histamine in less-than-fresh fish serves as a toxicological alert. Consequently, the analytical assessment of parvalbumin and histamine levels in fish becomes a critical public health safety measure. The multiplex detection of both analytes has emerged as an important issue. The analytical detection of parvalbumin and histamine requires different assays; while the determination of parvalbumin is commonly carried out by enzyme-linked immunosorbent assay, histamine is analyzed by high-performance liquid chromatography. In this study, we present an approach for multiplexing detection and quantification of trace amounts of parvalbumin and histamine in canned fish. This is achieved through a colorimetric and surface-enhanced Raman-scattering-based competitive lateral flow assay (SERS-LFIA) employing plasmonic nanoparticles. Two distinct SERS nanotags tailored for histamine or ß-parvalbumin detection were synthesized. Initially, spherical 50 nm Au@Ag core-shell nanoparticles (Au@Ag NPs) were encoded with either rhodamine B isothiocyanate (RBITC) or malachite green isothiocyanate (MGITC). Subsequently, these nanoparticles were bioconjugated with anti-ß-parvalbumin and antihistamine, forming the basis for our detection and quantification methodology. Additionally, our approach demonstrates the use of SERS-LFIA for the sensitive and multiplexed detection of parvalbumin and histamine on a single test line, paving the way for on-site detection employing portable Raman instruments.

SERS sensing for cancer biomarker: Approaches and directions.

These days, cancer is thought to be more than just one illness, with several complex subtypes that require different screening approaches. These subtypes can be distinguished by the distinct markings left by metabolites, proteins, miRNA, and DNA. Personalized illness management may be possible if cancer is categorized according to its biomarkers. In order to stop cancer from spreading and posing a significant risk to patient survival, early detection and prompt treatment are essential. Traditional cancer screening techniques are tedious, time-consuming, and require expert personnel for analysis. This has led scientists to reevaluate screening methodologies and make use of emerging technologies to achieve better results. Using time and money saving techniques, these methodologies integrate the procedures from sample preparation to detection in small devices with high accuracy and sensitivity. With its proven potential for biomedical use, surface-enhanced Raman scattering (SERS) has been widely used in biosensing applications, particularly in biomarker identification. Consideration was given especially to the potential of SERS as a portable clinical diagnostic tool. The approaches to SERS-based sensing technologies for both invasive and non-invasive samples are reviewed in this article, along with sample preparation techniques and obstacles. Aside from these significant constraints in the detection approach and techniques, the review also takes into account the complexity of biological fluids, the availability of biomarkers, and their sensitivity and selectivity, which are generally lowered. Massive ways to maintain sensing capabilities in clinical samples are being developed recently to get over this restriction. SERS is known to be a reliable diagnostic method for treatment judgments. Nonetheless, there is still room for advancement in terms of portability, creation of diagnostic apps, and interdisciplinary AI-based applications. Therefore, we will outline the current state of technological maturity for SERS-based cancer biomarker detection in this article. The review will meet the demand for reviewing various sample types (invasive and non-invasive) of cancer biomarkers and their detection using SERS. It will also shed light on the growing body of research on portable methods for clinical application and quick cancer detection.

Health and wellbeing status of the long-lived individuals of the Spanish LONGECYL cross-sectional study.

BACKGROUND: The increase in life expectancy and long-lived individuals is a challenge for public health and provides an opportunity to understand the determinants of longevity. However, few studies have addressed the factors associated with the health status and quality of life in a long-lived individual population. We described the perceived health, clinical status, quality of life, and dependency for activities of daily living in a representative population in Castile and Leon, Spain. METHODS: A sample of 759 long-lived individuals aged 95 years and older was studied by the Health Sentinel Network of Castile and Leon (Spain) through a health examination and a structured questionnaire covering quality of life (EQ-5D-3), lifestyle habits, diet, working life and family health. A blood sample was taken for the study of biological and genetic markers. Chi Square and logistic regression OR with 95% confidence intervals were used to analyze the determinants of the long-lived individuals' health status. The significant level for the bivariate analysis was established at 0.05. RESULTS: Perceived health was good, very good or excellent in 64.2%, while only 46.0% had a quality-of-life index above 0.5 (ranging from 0 to 1) and 44.1% maintained acceptable independence for activities of daily living. Quality-of-life index was higher in the oldest, (OR 7.98 [2,32-27.41]) above 100 years compared to those under 98, and men had better values for independence than women (OR 2.43 [1.40-4.29]). Cardiovascular diseases were the most prevalent (85.5%), but neurological and mental diseases and vision problems had the highest impact on quality of life and independence. CONCLUSION: The long-lived individuals of Castile and Leon have a relatively well-preserved health status, although the perception of health is higher than that describing their quality of life and dependence. The quality of life was higher in the oldest age group and showed differences according to sex, with a better quality of life in men. Public health policies and programs should take in account the differences by sex and age as well as the prevention and control of the main conditions related with poor quality of life or dependence. Future research must include the interaction among genetic, socioeconomic, environmental, and other clinical factors in the quality of life and disability of long-lived individuals.

Preoperative serum CA 72.4 as prognostic factor of recurrence and death, especially at TNM stage II, for colorectal cancer.

BACKGROUND: Nowadays, evaluation of colorectal cancer prognosis and decision-making for treatment continues to be based primarily on TNM tumour stage. Administration of adjuvant chemotherapy is especially challenging for stage II patients that can have very different disease-related outcomes. Therefore, more reliable prognostic markers need to be developed to improve the selection of stage II patients at high risk for recurrence. Our purpose is to assess the prognostic value of preoperative serum CA 72.4 to improve the risk stratification of CRC patients. METHODS: Preoperative sera collected from 71 unselected patients between January 1994 and February 1997 was assayed for CA 72.4 and CEA levels. Patients were followed-up for at least 30 months or until relapse. Survival curves were estimated by the Kaplan-Meier method and the prognostic value was determined using Log-Rank test and Cox regression analysis. RESULTS: Preoperative CA 72.4 levels above 7 U/mL correlate with a worse prognosis, with associated recurrence and death percentages exceeding the displayed by CEA. In a multivariate analysis, its combination with CEA proved the most important independent factor predicting survival. Remarkably, at stage II CA 72.4 also discriminates better than CEA those patients that will relapse or die from those with a favourable prognosis; however, CEA has not a negligible effect on survival. CONCLUSIONS: The most outstanding finding of the present work is the correct classification of nearly every patient with bad prognosis (relapse or death) at TNM stage II when CEA and CA 72.4 are used altogether. This could improve the decision-making involved in the treatment of stage II colon cancer. Certainly further large-scale studies must be performed to determine whether CA 72.4 can be effectively used in the clinical setting.

Decreased expression of alpha-L-fucosidase gene FUCA1 in human colorectal tumors.

In previous studies we described a decreased alpha-L-fucosidase activity in colorectal tumors, appearing as a prognostic factor of tumoral recurrence. The aim of this work was to extend the knowledge about tissue alpha-L-fucosidase in colorectal cancer by quantifying the expression of its encoding gene FUCA1 in tumors and healthy mucosa. FUCA1 mRNA levels were measured by RT-qPCR in paired tumor and normal mucosa tissues from 31 patients. For the accuracy of the RT-qPCR results, five candidate reference genes were validated in those samples. In addition, activity and expression of alpha-L-fucosidase in selected matched tumor and healthy mucosa samples were analyzed. According to geNorm and NormFinder algorithms, RPLP0 and HPRT1 were the best reference genes in colorectal tissues. These genes were used for normalization of FUCA1 expression levels. A significant decrease of more than 60% in normalized FUCA1 expression was detected in tumors compared to normal mucosa (p = 0.002). Moreover, a gradual decrease in FUCA1 expression was observed with progression of disease from earlier to advanced stages. These findings were confirmed by Western blot analysis of alpha-L-fucosidase expression. Our results demonstrated diminished FUCA1 mRNA levels in tumors, suggesting that expression of tissue alpha-L-fucosidase could be regulated at transcriptional level in colorectal cancer.

Geriatric assessment in hematology scale predicts treatment tolerability in older patients diagnosed with hematological malignancies: The RETROGAH study.

INTRODUCTION: The GAH (Geriatric Assessment in Hematology) scale is a psychometrically valid tool aimed at identifying older patients with hematological malignancies at higher risk of treatment-related toxicity. Our objective in this study was to determine the weights for each dimension of the GAH scale and the cut-off point to reliably predict treatment tolerability in this population, estimated by a weighted receiver operating characteristic (ROC) analysis and quantified by the area under the curve (AUC). MATERIAL AND METHODS: The RETROGAH was a retrospective cohort study including 126 patients who had previously participated in the GAH study. Patients were ≥ 65 years old with newly diagnosed myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML), multiple myeloma (MM), or chronic lymphoid leukemia (CLL) and treated with standard front-line therapy within three months after having completed the GAH scale. RESULTS: The optimal cut-off value of the GAH total score to discriminate patients at higher risk of treatment toxicity was 42, with 68.5% sensitivity and 55.8% specificity. Using this value, 66.1% of patients evaluated were found to develop some type of toxicity. The AUC was 0.6259 (95% CI: 0.512-0.739; p = 0.035). DISCUSSION: The GAH scale not only would enable clinicians to individualize therapy based on individual risk of toxicity but also discriminate patients that will benefit most from intensive treatments from those requiring an adapted approach. While futures studies in clinical practice may improve the model and overcome its limitations, the GAH scale should not be used alone when making treatment decisions.

Impact of an educational intervention on the quality of the Mediterranean diet, physical activity and weight status in adolescents: Randomised clinical trial. / Impacto de una intervención educacional sobre la calidad de la dieta mediterránea, la actividad física y el estado ponderal en adolescentes: ensayo clínico aleatorizado.

OBJECTIVE: To analyse the impact of an educational intervention on the quality of the Mediterranean diet, physical activity and weight status in adolescents. METHOD: Randomised clinical trial (RCT), controlled with a multimodal educational intervention (control group [n=36] and experimental group [n=46]). Data collection at the beginning and end of the study, in teenagers from Cáceres, Spain. In both groups anthropometric measurements and sociodemographic data were determined. The quality of the Mediterranean diet was assessed through the KIDMED test, the degree of physical activity through the PAQ-A questionnaire and weight status with the growth charts of the Faustino Orbegozo Eizaguirre Foundation. RESULTS: We obtained a significant increase in the experimental group in the PAQ-A questionnaire that assesses physical activity (P=.029). No significant differences were observed between groups in the weight status (P=.916). When comparing the quality of the Mediterranean diet (high vs moderate or low quality) with physical activity (Pcontrol=.730; experimental P=.495) and with weight status (Pcontrol=.838; experimental P=.372), No significant differences are observed. CONCLUSIONS: The educational intervention did not improve the quality of the Mediterranean diet or physical activity, although most of our sample had normal weight and acceptable physical activity. We must continue to improve the healthy eating pattern of our adolescents, to ensure an adequate state of health in the future.

A rapid calprotectin test for the diagnosis of pleural effusion.

In previous studies, measuring the levels of calprotectin in patients with pleural effusion (PE) was an exceptionally accurate way to predict malignancy. Here, we evaluated a rapid method for the measurement of calprotectin levels as a useful parameter in the diagnosis of malignant pleural effusion (MPE) in order to minimise invasive diagnostic tests. Calprotectin levels were measured with Quantum Blue® sCAL (QB®sCAL) and compared with the gold standard reference ELISA method. Calprotectin levels in patients with benign pleural effusion (BPE) were significantly higher (p < 0.0001) than for MPE patients. We measured the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and positive and negative likelihood ratios (LRs) for a cut-off value of ≤ 14,150 ng/mL; the diagnostic accuracy was 64%. The odds ratio for PE calprotectin levels was 10.938 (95% CI [4.133 - 28.947]). The diagnostic performance of calprotectin concentration was better for predicting MPE compared to other individual parameters. Comparison of two assays showed a slope of 1.084, an intercept of 329.7, and a Pearson correlation coefficient of 0.798. The Bland-Altman test showed a positive bias for the QB®sCAL method compared to ELISA fCAL®. Clinical concordance between both these methods was 88.5% with a Cohen kappa index of 0.76 (95% CI [0.68 - 0.84]). We concluded that QB®sCAL is a fast, reliable, and non-invasive diagnostic tool for diagnosing MPE and represents an alternative to ELISA that could be implemented in medical emergencies.

Avian reovirus sigmaA localizes to the nucleolus and enters the nucleus by a nonclassical energy- and carrier-independent pathway.

Avian reovirus sigmaA is a double-stranded RNA (dsRNA)-binding protein that has been shown to stabilize viral core particles and to protect the virus against the antiviral action of interferon. To continue with the characterization of this viral protein, we have investigated its intracellular distribution in avian cells. Most sigmaA accumulates into cytoplasmic viral factories of infected cells, and yet a significant fraction was detected in the nucleolus. The protein also localizes in the nucleolus of transfected cells, suggesting that nucleolar targeting is not facilitated by the viral infection or by viral factors. Assays performed in both intact cells and digitonin-permeabilized cells demonstrate that sigmaA is able to enter the nucleus via a nucleoporin-dependent nondiffusional mechanism that does not require added cytosolic factors or energy input. These results indicate that sigmaA by itself is able to penetrate into the nucleus using a process that is mechanistically different from the classical nuclear localization signal/importin pathway. On the other hand, two sigmaA arginines that are necessary for dsRNA binding are also required for nucleolar localization, suggesting that dsRNA-binding and nucleolar targeting are intimately linked properties of the viral protein.

Validation of Calprotectin As a Novel Biomarker For The Diagnosis of Pleural Effusion: a Multicentre Trial.

Discriminating between malignant pleural effusion (MPE) and benign pleural effusion (BPE) remains difficult. Thus, novel and efficient biomarkers are required for the diagnosis of pleural effusion (PE). The aim of this study was to validate calprotectin as a diagnostic biomarker of PE in clinical settings. A total of 425 patients were recruited, and the pleural fluid samples collected had BPE in 223 cases (53.7%) or MPE in 137 patients (33%). The samples were all analysed following the same previously validated clinical laboratory protocols and methodology. Calprotectin levels ranged from 772.48 to 3,163.8 ng/mL (median: 1,939 ng/mL) in MPE, and 3,216-24,000 ng/mL in BPE (median: 9,209 ng/mL; p < 0.01), with an area under the curve of 0.848 [95% CI: 0.810-0.886]. For a cut-off value of ≤ 6,233.2 ng/mL, we found 96% sensitivity and 60% specificity, with a negative and positive predictive value, and negative and positive likelihood ratios of 96%, 57%, 0.06, and 2.4, respectively. Multivariate analysis showed that low calprotectin levels was a better discriminator of PE than any other variable [OR 28.76 (p < 0.0001)]. Our results confirm that calprotectin is a new and useful diagnostic biomarker in patients with PE of uncertain aetiology which has potential applications in clinical practice because it may be a good complement to cytological methods.

Crystal structure of the avian reovirus inner capsid protein sigmaA.

Avian reovirus, an important avian pathogen, expresses eight structural and four nonstructural proteins. The structural sigmaA protein is a major component of the inner capsid, clamping together lambdaA building blocks. sigmaA has also been implicated in the resistance of avian reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed sigmaA by molecular replacement and refined it using data to 2.3-A resolution. Twelve sigmaA molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of sigmaA on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of sigmaA to double-stranded RNA. The minimal length of double-stranded RNA required for sigmaA binding was observed to be 14 to 18 bp.