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Introduction Serum neurofilament light chain (sNfL) levels are biomarkers of neuro-axonal injury in multiple neurological diseases. Little is known on their potential role as prognostic markers in people without known neurological conditions. Objective The aim of this study is to evaluate the association between sNfL levels and all-cause mortality in a general population setting. Methods sNfL levels were measured in 2071 people aged 25–75 years from the general US population that participated in the 2013–2014 cycles of the National Health and Nutrition Examination Survey (NHANES). Cognitive function was evaluated in a subset of participants aged 60–75 years using the Consortium to Establish a Registry for Alzheimer’s Disease-Word Learning test, the Animal Fluency test and the Digit Symbol Substitution test. We applied Cox proportional hazard models adjusted for several potential confounders to evaluate the association between sNfL and all-cause mortality through December 2019 by linking NHANES data with data from the National Death Index. Results In a cross-sectional analysis, higher sNfL levels were associated with worse performance in all three cognitive function tests. Over a median follow-up of 6.1 years, 85 participants died. In a multivariable model adjusted for age, sex, race-ethnicity, diabetes, chronic kidney disease, harmful alcohol consumption, cigarette smoke and prevalent cardiovascular disease, higher sNfL levels were significantly and positively associated with all-cause mortality (HR per unit increase in log-transformed sNfL: 2.46, 95% CI 1.77–3.43, p Conclusion We found a positive association between sNfL levels and mortality in the general US population. Further studies are needed to understand the biological mechanisms underlying this association.

Context Serum neurofilament light chain (sNfL) levels are biomarkers of neuroaxonal injury in multiple neurological diseases. Objective Given the paucity of data on the distribution of sNfL levels in the general population, in the present study we identified predictors of sNfL levels in a community setting and investigated the association between diabetes and sNfL. Methods sNfL levels were measured in 2070 people aged 20 to 75 years from the general US population (275 with and 1795 without diabetes) that participated in the 2013-2014 cycle of the National Health and Nutrition Examination Survey. We evaluated the association between diabetes and sNfL levels after adjustment for age, sex, race-ethnicity, alcohol use, and kidney function using a multivariable linear regression model. Cognitive function was evaluated in a subset of participants aged 60 to 75 years using the Consortium to Establish a Registry for Alzheimer's Disease-Word Learning test, the Animal Fluency test, and the Digit Symbol Substitution test. Results The weighted prevalence of diabetes was 10.4% (95% CI, 9.0-11.9). In each age stratum, patients with diabetes exhibited higher sNfL levels compared with nondiabetic participants. Age, proportion of males, prevalence of diabetes, and homeostatic model of insulin resistance increased progressively across quartiles of sNfL levels in the overall population, whereas estimated glomerular filtration rate (eGFR) showed an opposite trend. In the multivariable model, age, sex, eGFR, alcohol use and diabetes were significantly associated with sNfL levels. Moreover, higher sNfL levels were associated with worse performance in all 3 cognitive function tests. Conclusion Diabetes is associated with higher sNfL. Further large-scale and prospective studies are needed to replicate our results and evaluate the ability of sNfL to predict the incidence of neuropathy and dementia in this patient population.

Context Laparoscopic sleeve gastrectomy (LSG) is a recognized effective procedure of bariatric surgery, but a poor response in weight loss may still represent a clinical problem. To date there are no validated predictors useful to better perform patient selection. Objective To establish the association of baseline anthropometric, metabolic, and psychologic features with the percent total weight loss (%TWL) and percent excess weight loss (%EWL) 12 months after surgery. Design Retrospective longitudinal analysis of a set of data about obese patients attending the outpatient service of a single obesity center from June 2016 to June 2019. Patients A total of 106 obese patients underwent LSG with presurgery evaluation and follow-up at 12 months after surgery. Main outcome Weight loss 12 months after LSG. Results Patients who achieved a %TWL higher than the observed median (≥34%) were younger, with a lower fasting plasma glucose and glycated hemoglobin, with a lower prevalence of hypertension and with a lower score in the impulsiveness scale, compared with patients with a %TWL Conclusion Metabolic and psychologic features at baseline were independently associated with weight loss and explained a non-negligible effect on the response to LSG. These data suggest that careful metabolic and psychologic profiling could help in sharper indications and personalized pre- and postsurgical follow-up protocols in candidates for LSG.

Purpose The purpose of this study was to evaluate the impact of pre-existing diabetes on in-hospital mortality in patients admitted for Coronavirus Disease 2019 (COVID-19). Methods This is a single center, retrospective study conducted at Policlinico di Monza hospital, located in the Lombardy region, Northern Italy. We reviewed medical records of 373 consecutive adult patients who were hospitalized with COVID-19 between February 22 and May 15, 2020. Data were collected on diabetes status, comorbid conditions and laboratory findings. Multivariable logistic regression was performed to evaluate the effect of diabetes on in-hospital mortality after adjustment for potential confounding variables. Results Mean age of the patients was 72 ± 14 years (range 17–98), 244 (65.4%) were male and 69 (18.5%) had diabetes. The most common comorbid conditions were hypertension (237 [64.8%]), cardiovascular disease (140 [37.7%]) and malignant neoplasms (50 [13.6%]). In-hospital death occurred in 142 (38.0%) patients. In the multivariable model older age (Relative Risk [RR] 1.06 [1.04–1. 09] per year), diabetes (RR 1.56 [1.05–2.02]), chronic obstructive pulmonary disease (RR 1.82 [1.13–2.35]), higher values of lactic dehydrogenase and C-reactive protein were independently associated with in-hospital mortality. Conclusion In this retrospective single-center study, diabetes was independently associated with a higher in-hospital mortality. More intensive surveillance of patients with this condition is to be warranted.

Context Growing evidence suggests that appropriate levothyroxine (LT4) replacement therapy may not correct the full set of metabolic defects afflicting individuals with hypothyroidism. Objective To assess whether obese subjects with primary hypothyroidism are characterized by alterations of the resting energy expenditure (REE). Design Retrospective analysis of a set of data about obese women attending the outpatients service of a single obesity center from January 2013 to July 2019. Patients A total of 649 nondiabetic women with body mass index (BMI) > 30 kg/m2 and thyrotropin (TSH) level 0.4–4.0 mU/L were segregated into 2 groups: patients with primary hypothyroidism taking LT4 therapy (n = 85) and patients with normal thyroid function (n = 564). Main outcomes REE and body composition assessed using indirect calorimetry and bioimpedance. Results REE was reduced in women with hypothyroidism in LT4 therapy when compared with controls (28.59 ± 3.26 vs 29.91 ± 3.59 kcal/kg fat-free mass (FFM)/day), including when adjusted for age, BMI, body composition, and level of physical activity (P = 0.008). This metabolic difference was attenuated only when adjustment for homeostatic model assessment of insulin resistance (HOMA-IR) was performed. Conclusions This study demonstrated that obese hypothyroid women in LT4 therapy, with normal serum TSH level compared with euthyroid controls, are characterized by reduced REE, in line with the hypothesis that standard LT4 replacement therapy may not fully correct metabolic alterations related to hypothyroidism. We are not able to exclude that this feature may be influenced by the modulation of insulin sensitivity at the liver site, induced by LT4 oral administration.

Significance Thanks to their potent built-in adjuvanticity, bacterial outer membrane vesicles (OMVs) represent an attractive vaccine platform. However, their full-blown exploitation relies on the availability of efficient engineering strategies. This work provides strong experimental evidence that OMVs can be successfully decorated with heterologous antigens by channeling them to the lipoprotein transport machinery. Not only did lipidated antigens accumulate in the vesicular compartment at high levels but they also interfered with the acylation process of lipid A, thus substantially reducing the lipopolysaccharide-mediated reactogenicity, a major hurdle for vaccine applications. The approach was validated with 5 Staphylococcus aureus antigens, and mice immunized with engineered OMVs were completely protected against S. aureus challenge, paving the way for development of vaccines against this important pathogen., Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 μg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics.

Context It is still debated whether nonalcoholic fatty liver disease (NAFLD) may be a risk factor for reduced bone mineral density (BMD), and it is not known whether liver fibrosis, the major predictor of future development of liver-related events in NAFLD, has an influence on BMD. Objective To assess whether liver steatosis and fibrosis are associated with reduced BMD in the general US population. Methods We performed a cross-sectional analysis of the population-based 2017–2018 cycle of the National Health and Nutrition Examination Survey (NHANES), in which vibration-controlled transient elastography (VCTE) and dual-energy x-ray absorptiometry (DXA) of the femoral neck were simultaneously available. Controlled attenuation parameter (CAP) ≥ 274 dB/m was considered indicative of liver steatosis, while a median liver stiffness measurement (LSM) ≥ 8 kPa indicated the presence of significant liver fibrosis. We included all participants older than 50 years with reliable VCTE and femoral neck DXA results (925 men and 859 women). The main outcome measures were femoral neck BMD values indicative of osteopenia or osteoporosis. Results Steatosis and significant fibrosis were highly prevalent in the studied population, being present in 53.1% and 9.6% of men and 44.2% and 8.0% of women, respectively. In univariate analysis, liver steatosis was associated with a lower prevalence of osteoporosis in both men and women, while no difference was noted according to the degree of liver fibrosis. After adjustment for potential confounders, including age, BMI, race/ethnicity, cigarette smoking, and diabetes, neither CAP nor LSM were significantly associated with reduced BMD in both sexes. Conclusion Liver steatosis and fibrosis are not associated with femoral DXA-based diagnosis of osteopenia or osteoporosis in the US population older than 50 years.

Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines.

Aims: Hypothalamus-pituitary-adrenal (HPA) axis hyperactivity was suggested to be associated with the metabolic syndrome (MS), obesity and diabetes. The aim of this study was to test whether hypercortisolism was associated with altered glucose homeostasis and insulin resistance, hypertension and dyslipidemia in a homogeneous population of obese patients. Materials/Methods: In retrospective analysis of a set of data about obese patients attending the outpatient service of a single obesity centre between January 2013 and January 2020, 884 patients with BMI >30 kg/m2 were segregated in two subgroups: patients with urinary free cortisol (UFC) higher than normal (UFC+; n = 129) or within the normal range (UFC−; n = 755). Results: The overall prevalence of UFC+ was 14.6% and double test positivity (morning cortisol >1.8 mcg/dL following overnight dexamethasone suppression test, ODST) was detected in 1.0% of patients. Prediabetes (OR 1.74; 95%CI 1.13-2.69; p = 0.012) and diabetes (OR 2.03; 95%CI 1.21-3.42; p = 0.008) were associated with higher risk of UFC+ when analysis was adjusted for confounding variables. Conversely, hypertension and dyslipidemia were not related to UFC+. Within the individuals with normal FPG and HbA1c, those with higher estimated insulin resistance (HOMA2-IR) maintained a higher risk of UFC+ (OR 2.84, 95%CI 1.06-7.63; p = 0.039) and this relationship was weakened only when the body fat percentage was included into the model. Conclusions: In obese patients, hypercortisolism was more frequent across the entire spectrum of altered glucose homeostasis including the very early stages; this relation could not be detected for the other criteria of the MS, as waist, hypertension and atherogenic dyslipidemia.

Background and aims Seasonal variations in several risk factors for cardiovascular events (CVD) were described. Here, we evaluate the impact of seasonal variations in blood pressure (BP), lipid profile and glycemic control on estimated CVD risk in patients with type 2 diabetes (T2D). Methods and results Retrospective monocentric study of patients with T2D who were visited at least once in the winter period and once in the summer period, less than 8 months apart, for which data related to systolic (S) BP, diastolic (D) BP, body mass index, glycosylated hemoglobin (HbA1c), total cholesterol, HDL cholesterol and smoking habit were available on both occasions. The 10-year CVD risk was calculated using the UKPDS risk engine and the ASCVD risk estimator. As many as 411 patients were included in the study. Significant within-patient differences between summer and winter were found for the absolute risk of events assessed with both calculators (Δs-w UKPDS-CHD: −1.33%, Δs-w UKPDS-Stroke: −0.84%, Δs-w ASCVD: −2.21%). The seasonal change in SBP was the main responsible for the change in risk estimated with both the UKPDS-Stroke (r2 = 0.43) and the ASCVD (r2 = 0.50) scores, while the change in total cholesterol was the main determinant of the change in risk for the UKPDS-CHD (r2 = 0.34). A significant correlation was identified between changes in temperature and changes in SBP (ρ = 0.130, p = 0.008), but not in other risk factors. Conclusions Seasonal variations in the classic CVD risk factors influence the risk estimated using validated calculators.

Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines.

Background: Bariatric surgery is a valuable therapeutic option in the treatment of obesity but the outcomes show a large subject-to-subject variability yet to be explained. Thyroid function may represent an involved factor and we have only few controversial data about its influence. Subjects/methods: We retrospectively assessed using a longitudinal approach the relation between baseline TSH levels and short-term (6 and 12 months) weight loss in 387 euthyroid patients who underwent laparoscopic gastric banding (LAGB; n = 187) or sleeve gastrectomy (SG; n = 200). Results: After LAGB, patients with low-normal TSH levels (0.40–1.40 mUI/L) had higher percent total weight loss, ∆BMI and percent excess weight loss when compared to patients with normal (1.41–2.48 mUI/L) and high-normal (2.49–4.00 mUI/L) TSH (p < 0.05). Conversely, no association was detected after SG (p = 0.17). The multivariable regression analysis showed that also baseline BMI (6–12 months) and HOMA2-IR (only at 6 months) were independently associated with the outcomes. Conclusions: TSH levels may influence the short-term weight loss response after LAGB. The lack of association after SG suggests that the influence of baseline endocrine and metabolic factors may not be relevant for procedures with greater and more immediate calorie intake restriction.

Objective: Visit-to-visit variability in SBP is a risk factor for cardiovascular disease (CVD) in type 2 diabetes (T2DM) but little is known on whether in T2DM this differs according to presence or absence of previous CVD. Methods: We retrospectively assessed the coefficient of variation (standard deviation/mean) of mean SBP (SBP-CV) in 970 patients (44% with established CVD) attending at least four times our secondary care diabetes clinic in 2015–2016 to estimate their risk of CVD-related events using the 10-year UKPDS Risk Engine. Results: Patients with established CVD had a higher SBP-CV (10.3 ± 4.8%) than patients without CVD (8.9 ± 4.3%; P < 0.001) as confirmed by the progressively higher prevalence of established CVD in tertiles of SBP-CV (36.6, 46.1, and 52.0%; P < 0.001), in association with more aggressive and complex drug regimens. On the basis of the 10-year UKPDS Risk Engine, higher SBP-CV values were associated with increased risks of the CVD outcomes regardless of the previous history of CVD in multivariate models. Conclusion: Visit-to-visit variability of SBP was greater in T2DM patients with that in those without previous history of CVD, and maintained an independent association with higher estimated risk of CVD-related events regardless of the history of CVD, suggesting that its prognostic significance is relevant in the entire CVD continuum of patients with T2DM.

ObjectiveNon-alcoholic fatty liver disease (NAFLD) is prevalent in patients with type 2 diabetes. Here, we estimate the proportion of patients with type 2 diabetes that should be referred to hepatologists according to the European Association for the Study of the Liver (EASL)-European Association for the Study of Diabetes (EASD)-European Association for the Study of Obesity (EASO) Guidelines and evaluate the association between non-invasive biomarkers of steatosis and fibrosis and diabetic complications.Research design and methodsThis is a retrospective analysis of type 2 diabetes patients who attended on a regular basis our diabetes clinic between 2013 and 2018 (n=2770). Steatosis was assessed using Fatty Liver Index (FLI), Hepatic Steatosis Index and NAFLD Ridge Score and fibrosis using NAFLD Fibrosis Score (NFS), Fibrosis-4 (FIB-4), aspartate aminotransferase (AST) to platelet ratio index (APRI) and AST/alanine aminotransferase (ALT) ratio. Outcome measures were altered albumin excretion rate (AER), chronic kidney disease (CKD) and cardiovascular disease (CVD).ResultsThe prevalence of advanced fibrosis varied from 1% (APRI) to 33% (NFS). The application of the guidelines using a sequential combination of FLI and FIB-4 would lead to referral of 28.3% of patients when using standard FIB-4 cut-offs, while this number dropped to 13.4% when age-adjusted FIB-4 thresholds were applied. A higher prevalence of altered AER was associated with liver steatosis (FLI: OR: 3.49; 95% CI 2.05 to 5.94, pConclusionsWhile specific fibrosis scores identify different proportion of patients with advanced fibrosis, the use of age-adjusted FIB-4 cut-offs leads to a drop in gray-zone results, making referrals to hepatologists more sustainable. Interestingly non-invasive biomarkers were consistently associated with a different pattern of diabetic complications.

Iodine deficiency still remains a significant health issue worldwide. Pregnant and lactating women are at risk for iodine deficiency when living in mild iodine-deficient areas such as Italy. This study aims at evaluating the consumption of iodized salt, iodine-rich-foods and maternal micronutrient supplements in a group of women with limited access to the Italian National Health System. A cross-sectional survey was conducted among immigrant and Italian women living in poverty and referring to 40 Non-Governmental Organization throughout Italy for their health needs. 3483 women answered the ad hoc questionnaire between January 2017 and February 2018. The consumption of iodized salt was very low, and even lower among immigrant women. Determinants of iodized salt consumption were the period spent in Italy for immigrant women and living in a family-type setting, parity and, particularly, the degree of education for Italian ones. 17.5% of immigrant women and 8.6% of the Italian ones reported a diagnosis of thyroid disease. 521 women, 75.4% of whom were immigrants, were pregnant or breast-feeding. The majority (57.3%) had no specific maternal supplementation. Both Italian and immigrating women with a low income or without access to the public health system have a poor adherence both to the salt iodization policy and to folic acid and iodine supplements in preconception and pregnancy. They also referred a low-frequency intake of iodine-rich-foods. The identification of barriers to health care access could be useful to promote specific health interventions in this target population.

Purpose: The purpose of this study was to estimate how many individuals with severe obesity and NAFLD should be referred to hepatologists according to the EASL–EASD–EASO guidelines and whether the choice of specific indicators of liver fibrosis would significantly impact the number of referrals. Methods: This was a single-center retrospective study of 495 individuals with severe obesity screened at our institution between 2012 and 2018 for a bariatric surgery intervention. The guidelines were applied using the NAFLD Liver Fat Score (NLFS) to assess the presence of steatosis and the NAFLD fibrosis score (NFS), Fibrosis-4 (FIB-4) and Hepamet Fibrosis Score (HFS) to assess the risk of advanced fibrosis. Results: Three hundred and seventy-nine patients (76.6%) had evidence of liver steatosis. The application of the guidelines would lead to referral of 66.3% of patients using NFS, 31.7% using FIB-4 and 34.2% using HFS. When referrals due to abnormal liver function tests were excluded, these percentages dropped to 55.8%, 7.3% and 12.1%, respectively. The strongest inter-biomarker agreement was found between FIB-4 and HFS (κ = 0.86, 95% CI 0.815–0.910). Conclusion: Strict application of the guidelines in individuals with severe obesity would probably lead to over-referral, although a great variability exists among the different scores.

Aim: To investigate the relative contribution of previous gestational diabetes mellitus (GDM) and current type 2 diabetes (T2D) on the development of liver fibrosis, the strongest predictor of end-stage liver disease. Methods: This is a population-based cross-sectional study based on data from the 2017–2018 cycle of the National Health and Nutrition Examination Survey. We included women age ≥ 20 years that had delivered at least one live birth and had available data on vibration-controlled transient elastography (VCTE). Liver steatosis and fibrosis were assessed by the median value of controlled attenuation parameter (CAP) and liver stiffness measurement (LSM), respectively. Results: Among the 1699 women included in the study, 144 (10.1%, 95% CI 7.7–13.2) reported a previous diagnosis of GDM. Women with previous GDM were younger, had a higher BMI, a higher prevalence of T2D and were significantly older at the time they had the last live birth. Univariate analysis did not show a significant difference between women with and without a prior history of GDM in terms of both steatosis (44.8% vs 39.4%, p = 0.464) and fibrosis (7.5% vs 7.6%, p = 0.854). Multivariable logistic regression analysis showed that BMI, γ-glutamyltranspeptidase levels, T2D (OR 2.96, 95% CI 1.48–5.93, p < 0.01), HBV and HCV infection were associated with higher odds of significant fibrosis, while previous GDM showed a neutral effect. Conclusions: Women with previous GDM that do not develop overt T2D might not experience a poor hepatic prognosis.

Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design vaccines with broad coverage. This approach opens a path to a new generation of vaccines. Tyrosine-ligation allows creation of more homogeneous vaccines, correlation of the immune response to defined connectivity points, and fine-tuning of the conjugation site in glycan-protein conjugates.

The aim of the study was to evaluate the diagnostic performance of a new ultrasound elastography (USE) parameter based on the measurement of the percentage of maximal stiffness within a nodule as compared with the already established elastographic strain index (SI) and to investigate their diagnostic performance according to nodule size.The study included 218 nodules. Each nodule underwent conventional ultrasound (US), USE evaluation, and fine-needle aspiration cytology (FNAC). Thyroid nodules were further stratified into 4 subgroups (G) according to their size (G1,1 cm; G2, 1-2 cm; G3,3 cm). USE evaluation comprised the measurement of the percentage of the areas included in the region of interest corresponding to the maximal stiffness (% Index) and of the SI.The % Index and of the SI were significantly higher in malignant than in benign thyroid nodules, and both measurements displayed a good diagnostic performance (SI sensitivity and specificity, 0.66 and 0.90, respectively; % Index sensitivity and specificity, 0.76 and 0.89, respectively). Compared with SI, the % Index was more informative, both in the whole group of thyroid nodules (odds ratio [OR], 18.68; 95% confidence interval [CI], 6.06 to 63.49; P.0001 versus OR, 26.15; 95% CI, 8.01 to 102.87; P.0001, respectively) and in the G1 and G2 subgroups.The % Index is a stronger predictor of nodule malignancy than both the SI and the conventional US signs. This is particularly true in nodules smaller than 1 cm, which are more difficult to explore both by conventional US and FNAC.

Background The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions. Results We first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 “dispensable” genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions. Conclusions This work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0681-1) contains supplementary material, which is available to authorized users.

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides.

Objectives: Experimental evidences indicate that leptin is involved in the neuroinflammatory process sustaining multiple sclerosis (MS). However, the relationship between leptin and body fat, as assessed by body mass index (BMI), in MS was not previously evaluated. It was the aim of this study to compare serum leptin levels between patients with MS and healthy controls and to evaluate the possible relationship between circulating leptin levels and disease severity. Patients and Methods: Eighty-four MS patients and 57 sex-matched healthy volunteers were enrolled. Serum leptin levels were measured in all patients and controls. MS patients were stratified in 3 groups according to their degree of disability as assessed by the Expanded Disability Status Scale (EDSS). Patients were classified as having low (33 patients with an EDSS score Results: No significant differences in serum leptin levels and BMI were observed between patients and controls. In patients with MS, serum leptin levels were significantly correlated with BMI in those patients with low (R2 = 0.363; p < 0.001) and intermediate disability (R2 = 0.408; p < 0.001), but not in patients with a higher disability score (R2 = 0.064; p = 0.256). Conclusion: BMI, the major determinant of leptin level in physiological conditions, has a minor role in determining the serum levels of leptin in MS patients with a high EDSS score. Future longitudinal studies will be required in order to provide further insights into the regulation of leptin secretion in patients with MS.

The outcome of antithyroid drug (ATD) treatment for Graves disease (GD) is difficult to predict. In this study, we investigated whether male gender, besides other factors usually associated with a poor outcome of ATD treatment, may affect disease presentation and predict the response to medical treatment in subjects with GD.We studied 294 patients with a first diagnosis of GD. In all patients, ATD treatment was started. Clinical features, thyroid volume, and eye involvement were recorded at baseline. Serum levels of free thyroxine (FT4), free triiodothyronine (FT3), thyroid-stimulating hormone (TSH), and TSH-receptor antibodies (TRAb) were measured at baseline and during the follow-up. Treatment outcome (FT4, FT3, and TSH serum levels and further treatments for GD after ATD withdrawal) was evaluated.When compared to women, men showed a significantly larger thyroid volume and a higher family positivity for autoimmune diseases. During ATD, the mean serum levels of TSH, FT4, FT3, and TRAb did not differ between groups. Within 1 year after ATD discontinuation, relapse of hyperthyroidism was significantly more frequent in men than in women. Within the 5-year follow-up period, the prevalence of men suffering a late relapse was higher compared with that of women. The outcome at the end of the 5-year follow-up period was significantly associated with gender and TRAb levels at disease onset.Male patients with GD have a poorer prognosis when submitted to medical treatment with ATDs. A larger goiter at presentation and a stronger genetic autoimmune background might explain this gender difference in patients with GD.ATD = antithyroid drug FT3 = free triiodothyronine FT4 = free thyroxine GD = Graves disease GO = Graves orbitopathy RAI = radioiodine TRAb = thyroid-stimulating hormone-receptor antibody TSH = thyroid-stimulating hormone.

Autoimmune thyroid disease (AITD) has been reported in patients with multiple sclerosis (MS) receiving interferon-beta (IFN-β), but not in those receiving Glatiramer acetate (GA). CXCL10 is a chemokine playing a pathogenetic role in AITD and MS. Our aim was to evaluate the effects on CXCL10 secretion of IFN-β and GA, alone and in combination with TNF-α, in primary cultures of thyrocytes (PCT). Significant and dose-dependent secretions of CXCL10 were induced by IFN-β but not GA. TNF-α synergistically increased IFN-β induced CXCL10 secretion. These results may provide an explanation for the occurrence of AITD during IFN-β, but not during GA, treatment for MS.

The Merseburg triad (hyperthyroidism, goiter, and orbitopathy) characterizes classical description of Graves’ disease (GD). Aim of this observational, longitudinal study was to evaluate the current clinical features of newly diagnosed GD in Italy. In two Northern Italy centers (Varese and Pavia), 283 consecutive patients (211 women, 72 men; mean age 47.4 years) with newly diagnosed GD were recruited in the years 2010–2014. Diagnosis was based on established criteria, and thyroid volume was assessed by ultrasonography. A clinical severity score (CSS) to assess the overall disease severity was developed by grading each component of the Merseburg triad. At diagnosis, 45 % of patients had no goiter, and 30 % had a small goiter. The proportion of goitrous patients was much lower than in two Italian studies performed 20–30 years ago. Hyperthyroidism was subclinical in 16 % and mild in 29 % of patients, and Graves’ orbitopathy was present in 20 %, usually mild, and active in only 2.5 % of patients. Using the CSS, less than half (44 %) of the patients had severe GD, while 22 % had mild and 34 % moderate disease. CSS was associated with a significantly higher risk of poorly controlled hyperthyroidism at 6 months. In Italy, a relevant proportion of Graves’ patients at diagnosis have mild to moderate GD; about half of them have no goiter, slightly less than one-fifth have subclinical hyperthyroidism, and only 20 % have GO. Thus, the clinical phenotype of GD is milder than in the past, possibly due to both earlier diagnosis and treatment, and improved iodine nutrition.

Background Protocols of controlled ovarian hyper-stimulation (COH) require, as a crucial step, the identification of reliable predictors of ovarian reserve. Anti-Mullerian hormone (AMH) is one of the most reliable predictors of ovarian reserve but other factors including autoimmune thyroid diseases (ATD) have been associated with reduced fertility and poor COH outcome. Aim of the present study was to evaluate the relationship between ATD and AMH, and their role on the outcome of COH. Methods The study group included 288 sub-fertile euthyroid women aged less than 40 years attending a single center for Reproductive Medicine. Among them, 55 were ATD-positive and 233 ATD-negative. The serum levels of AMH, FSH, LH, estradiol (E2), and TSH were measured before COH. The ratio between serum E2 concentration on the day of oocytes pick-up and the total dose of administered recombinant FSH (r-FSH) (E2/r-FSH ratio) was calculated. Results The serum levels of AMH were significantly related to E2/r-FSH ratio, total dose of r-FSH and number of M II oocytes, both in ATD-positive and ATD-negative women. Within the low stratum of AMH levels, the presence of ATD did not further affect the outcome of COH. When the serum levels of AMH were in the high stratum, the presence of ATD significantly affected the E2/rFSH ratio, the total dose of r-FSH and the number of M II oocytes. Conclusions The probability of a poor response to COH is high, and independent from ATD, in women with low AMH serum levels. In women with a good ovarian reserve, as assessed by high AMH serum levels, the presence of ATD impairs the outcome of COH.

Thyroid cancer may express estrogen receptors (ERs) and various grades of peri-tumor inflammation. The aim of the study was to evaluate the expression of ERs in relation to the TNM stage and peri-tumor inflammatory infiltrate in differentiated thyroid cancers. 127 patients (109 females, 18 males) with differentiated thyroid cancer (T1 = 91, T2 = 18, T3 = 11, T4 = 7) were evaluated. In tumors and in the correspondent extra-tumor parenchyma, ERs expression was evaluated by immunohistochemistry. In 114 tumors and correspondent peri-tumor tissues, the presence of inflammatory infiltration was also recorded. ER-alpha expression was higher in clinical than in incidental tumors of the T1 subgroup (p = 0.037), and was associated with capsular invasion in T2 tumors (p < 0.0001). ER-beta expression was negatively associated with vascular invasion in T1 (p = 0.005) and T2 tumors (p = 0.015). No significant relationship between ERs expression and tumor phenotype emerged in T3 and T4 subgroups. Tumors without inflammatory cell infiltrate showed a higher expression of both ER-alpha (p = 0.035) and ER-beta (p = 0.026) than the ones with inflammatory infiltrate. The relationship between tumor phenotype and ERs expression did not vary in the presence or absence of peri-tumor inflammatory infiltration. ER-alpha positivity and ER-beta negativity are associated with a more aggressive phenotype in both T1 and T2 thyroid cancers, suggesting that tumor biology may be more relevant than tumor size for cancer risk assessment. Inflammatory status is also associated with ERs expression, but not with tumor growth or phenotype.

Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date, and their mechanisms of transpeptidation and regulation need to be further investigated. The available 3-dimensional structures of these enzymes reveal a typical sortase fold, except for the presence of a unique feature represented by an N-terminal highly flexible loop known as the "lid." This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an autoinhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work, we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme.

Group B Streptococcus pili are covalently linked structures assembled via a sortase-catalyzed transpeptidation mechanism involving specific residues and motifs. A sequence element containing a conserved glutamic acid, called the E-box, has been described to be involved in pilus formation. Although it is known that the glutamic acid is involved in stabilizing the internal isopeptide bonds, its role in pilus assembly still needs to be investigated. Using site-specific mutagenesis and complementation studies of knockout strains, we found that the E-box glutamic residue of the backbone and the major ancillary proteins is essential for pilus protein polymerization. NMR analysis revealed that the mutation of this residue seriously affected the folding of the protein. By contrast, the mutation of the lysine involved in the same isopeptide bond did not engender a structural destabilization, and the native fold was preserved. Moreover, molecular dynamics simulations on the E-box-containing domain of the backbone protein showed that the E-box glutamic acid is necessary to maintain the appropriate dryness of the domain core and that its mutation favors an unfolded state. The data provide the first direct evidence that the E-box has an additional and key role in maintaining the correct protein fold independently of isopeptide bond formation.

MicroRNAs are small non-coding RNAs that modulate gene expression at post-transcriptional level, playing a crucial role in cell differentiation and development. Recently, some reports have shown that a limited number of mammalian microRNAs are also involved in anti-viral defense. In this study, the analysis of the hepatitis B virus (HBV) genome by the computer program MiRanda led to the identification of seven sites that are potential targets for human liver microRNAs. These sites were found to be clustered in a 995-bp segment within the viral polymerase ORF and the overlapping surface antigen ORF, and conserved among the most common HBV subtypes. The HBV genomic targets were then subjected to a validation test based on cultured hepatic cells (HepG2, HuH-7 and PLC/PRF/5) and luciferase reporter genes. In this test, one of the selected microRNAs, hsa-miR-125a-5p, was found to interact with the viral sequence and to suppress the reporter activity markedly. The microRNA was then shown to interfere with the viral translation, down-regulating the expression of the surface antigen. Overall, these results support the emerging concept that some mammalian microRNAs play a role in virus-host interaction. Furthermore, they provide the basis for the development of new strategies for anti-HBV intervention. © 2011 The Author(s).

ObjectiveThyroid ultrasound (US) scan is a valuable tool for diagnosing thyroid diseases. In autoimmune thyroid disease (AITD), an hypoechoic pattern of the thyroid at US is related to circulating thyroid antibodies (Abs). The aim of this study was to evaluate the diagnostic accuracy of thyroid US for the detection of AITD in patients with morbid obesity.DesignThyroid US scans showing an hypoechoic pattern of the thyroid were collected from 105 morbid obese patients (body mass index (BMI) >40 kg/m2) and 105 non-obese patients (BMI≤30 kg/m2).ResultsA thyroid hypoechoic pattern at US was consistent with clinical/biochemical features of AITD in 90/105 (85.7%) non-obese patients and in 22/105 (20.9%) morbid-obese patients (PPConclusionsOur results suggest that i) morbid obesity may affect thyroid morphology, and ii) an hypoechoic pattern of the thyroid at US, a well-established parameter for diagnosing AITD, has a poor diagnostic accuracy when patients with morbid obesity are taken into account.