Covalent coupling of HIV-1 glycoprotein trimers to biodegradable calcium phosphate nanoparticles via genetically encoded aldehyde-tags.

The usage of antigen-functionalized nanoparticles has become a major focus in the field of experimental HIV-1 vaccine research during the last decade. Various molecular mechanisms to couple native-like trimers of the HIV-1 envelope protein (Env) onto nanoparticle surfaces have been reported, but many come with disadvantages regarding the coupling efficiency and stability. In this study, a short amino acid sequence ("aldehyde-tag") was introduced at the C-terminus of a conformationally stabilized native-like Env. The post-translational conversion of a tag-associated cysteine to formylglycine creates a site-specific aldehyde group without alteration of the Env antigenicity. This aldehyde group was further utilized for bioconjugation of Env trimers. We demonstrated that the low acidic environment necessary for this bioconjugation is not affecting the trimer conformation. Furthermore, we developed a two-step coupling method for pH-sensitive nanoparticles. To this end, we conjugated aldehyde-tagged Env with Propargyl-PEG3-aminooxy linker (oxime ligation; Step-one) and coupled these conjugates by copper-catalyzed azide-alkyne cycloaddition (Click reaction; Step-two) to calcium phosphate nanoparticles (CaPs) functionalized with terminal azide groups. CaPs displaying orthogonally arranged Env trimers on their surface (o-CaPs) were superior in activation of Env-specific B-cells (in vitro) and induction of Env-specific antibody responses (in vivo) compared to CaPs with Env trimers coupled in a randomly oriented manner. Taken together, we present a reliable method for the site-specific, covalent coupling of HIV-1 Env native-like trimers to the surface of nanoparticle delivery systems. This method can be broadly applied for functionalization of nanoparticle platforms with conformationally stabilized candidate antigens for both vaccination and diagnostic approaches. STATEMENT OF SIGNIFICANCE: During the last decade antigen-functionalized nanoparticles have become a major focus in the field of experimental HIV-1 vaccines. Rational design led to the production of conformationally stabilized HIV-1 envelope protein (Env) trimers - the only target for the humoral immune system. Various molecular mechanisms to couple Env trimers onto nanoparticle surfaces have been reported, but many come with disadvantages regarding the coupling efficiency and stability. In this paper, we describe a highly selective bio-conjugation of Env trimers to the surface of medically relevant calcium phosphate nanoparticles. This method maintains the native-like protein conformation and has a broad potential application in functionalization of nanoparticle platforms with stabilized candidate antigens (including stabilized spike proteins of coronaviruses) for both vaccination and diagnostic approaches.

Relación entre los niveles de compuestos volátiles sulfurados con los niveles de RANKL y OPG en pacientes con periodontitis crónica moderada o severa: estudio transversal / Relationship between the levels of volatile sulfur compounds with the levels of RANKL and OPG in patients with moderate or severe periodontitis: A cross sectional study

Antecedentes: El efecto de los Compuestos Volátiles Sulfurados (CVS) sobre los tejidos periodontales, específicamente a nivel del eje RANKL/OPG, no ha sido dilucidado y en la actualidad existe escasa literatura al respecto publicada. Objetivo: Evaluar si los CVS medidos en la cavidad oral de pacientes con periodontitis crónica moderada a severa se relacionan con la expresión de RANKL y OPG a nivel de fluido gingival crevicular (FGC). Método: Se realizó un estudio transversal con 71 pacientes derivados de la Unidad de Diagnóstico de la Clínica Odontológica Docente Asistencial de la Universidad de los Andes. Posterior a la realización de un examen periodontal completo se tomaron muestra de los niveles de CVS de la boca de los pacientes mediante un monitor de sulfuros y muestras de FGC para evaluar los niveles de RANKL y OPG mediante test de ELISA. Los datos obtenidos fueron analizados mediante test de correlación de Spearman. Resultados: Al evaluar la correlación de los niveles de CVS con los niveles de RANKL, OPG y la razón RANKL/OPG, se observó un R de 0.098 con un p value = 0.41; -0.084 con un p value= 0.48 y 0.067 con un p value = 0.57 respectivamente. Conclusiones: El presente estudio no pudo demostrar si existe una relación entre los niveles de CVS con la expresión de RANKL y OPG en el FGC de pacientes con periodontitis crónica.

Background: The effect of the Volatile Sulfur Compounds (VSC) on the periodontal tissues, specifically at the RANKL/OPG level has not been elucidate and there is little literature published on this subject. Aim: To explore if the VSC levels measured in the oral cavity of patients with moderate or severe chronic periodontitis are correlated with the expression of RANKL and OPG at the gingival fluid level (GF). Method: A cross-sectional study was conducted with 71 patients referred by the Universidad de los Andes’s dentistry diagnosis department. After undergoing full-mouth periodontal exam, levels of CVS were recorded using a sulfur monitor and then GF was extracted to assess RANKL and OPG levels with ELISA. Data was analyzed with Spearman correlation test. Results: The correlation (R) between the VSC and the GF levels was 0.098 with a p value = 0.41; -0.084 with a p value = 0.48 y 0.067 with a p value = 0.57 respectively. Conclusions: This study could not demonstrate if there is a relation between the VSC levels and the expression of RANKL and OPG in the GF in patients with chronic periodontitis.

Cerebral venous thrombosis: diagnostic accuracy of combined, dynamic and static, contrast-enhanced 4D MR venography.

BACKGROUND AND PURPOSE: MR including MRV is an established method to diagnose CVT. However, it remains unsettled which MR imaging modalities offer the highest diagnostic accuracy. We evaluated the accuracy of a combined, dynamic (1.5 seconds per dataset) and static (voxel size, 1.1 x 0.9 x 1.5 mm), contrast-enhanced MRV method (combo-4D MRV) relative to other established MR/MRV modalities. MATERIALS AND METHODS: A total of 39 patients with CVT (n = 20) and control subjects (n = 19) underwent combo-4D MRV, 2D TOF MRV, GRE imaging, and T2W imaging. For these modalities, diagnostic accuracy (ROCs) for CVT affecting 53 out of 234 predefined venous segments was determined. Sensitivity and specificity were separately calculated for different stages of CVT (acute/subacute/chronic). RESULTS: Combo-4D MRV showed the highest accuracy (AUC, 0.99 [95% CI, 0.97-1.0]; sensitivity, 97% [84%-100%]) for thrombosed dural sinuses. For all thrombosed segments including cortical veins, its sensitivity was best (76% [64%-84%]; AUC, 0.92 [0.88-0.96]), followed by TOF MRV (72% [59%-81%]; AUC, 0.93 [0.88-0.97]). Even for chronic CVT, it showed a relatively high sensitivity of 67% (30%-90%). For thrombosed cortical veins alone, GRE images achieved the highest sensitivity (66% [46%-81%]; AUC, 0.88 [0.78-0.97]). Specificities of all modalities ranged from 96% to 99%. CONCLUSIONS: Combo-4D MRV showed an excellent accuracy for the diagnosis of dural sinus thrombosis. The analysis of dynamic patterns of contrast enhancement in dural sinuses appeared useful to identify chronic thrombosis. To diagnose thrombosed cortical veins, GRE images should primarily be analyzed.

Polymorphous low-grade adenocarcinoma versus pleomorphic adenoma of minor salivary glands: resolution of a diagnostic dilemma by immunohistochemical analysis with glial fibrillary acidic protein.

OBJECTIVES: Differentiating polymorphous low-grade adenocarcinoma (PLGA) from pleomorphic adenoma (PA) in salivary gland biopsy specimens from the palatal region might be a diagnostic dilemma for the pathologist when tumors are cellular with minimal matrix material. Glial fibrillary acidic protein (GFAP), expressed by a number of cells in the mature central nervous system, is also expressed in tumors not generally considered to be of glial origin. PAs have previously been reported to strongly express GFAP. PLGAs have been examined for the expression of this protein only in small group studies with variable results. The objective of this study was to determine whether differential expression of GFAP in these 2 tumors could be diagnostically significant. STUDY DESIGN: A total of 42 PLGAs and 36 PAs, formalin-fixed and paraffin-embedded, were immunostained with rabbit polyclonal antibodies to GFAP. CNS tissue was used as a positive control. RESULTS: Results showed the 36 cases of PA to be strongly positive for GFAP. Of PLGAs, 31 were negative and 11 showed faint patchy reactivity in luminal cells. CONCLUSIONS: The results strongly support a role for GFAP as a diagnostic adjunct in the microscopic differential diagnosis of PLGA versus PA. This study is the largest investigation with consistent results to date addressing the application of this antibody to the diagnostic problem of PA versus PLGA.

Reassessment of ADHD in a historical cohort of children treated with stimulants in the period 1969-1989.

The operational diagnostic criteria for ADHD or HKD were not at hand in Denmark before 1994, when WHO's International Classification of Diseases -10 (ICD-10) was introduced. The criteria for ADHD/HKD were well known at our clinic, but when treating children with stimulants the criteria used clinically prior to 1994 were less specific than today. The aims of this paper are to examine the prevalence of ADHD in a historical cohort of children treated with stimulants during the period 1969-1989 and to reassess a wide range of comorbid disorders and sociodemographic characteristics in order to compare aspects of the given treatment with the modern recommendations. At our clinic 208 children (183 boys) were given stimulants during the period 1969-1989. Case records on these probands were reassessed retrospectively for DSM-IV criteria regarding ADHD and comorbid disorders as well as for characteristics of stimulant treatment. Sixty-five percent met full criteria for an ADHD diagnosis. Including Subthreshold ADHD as many as 81 % of the cohort were re-diagnosed with ADHD. Prevalence rates of conduct problems and anxiety disorder were similar to previous prospective ADHD studies. The use of stimulants during the period 1969-1989 were in accord with modern guidelines. This cohort is comparable to other clinically based ADHD cohorts as regards inattentive, hyperactive/impulsive symptoms, comorbidity, impairment, gender differences, intelligence and socio-economic status (SES).

Gilles de la Tourette syndrome in a child with congenital deafness.

We present the case of a 10-year-old boy, Sam, with congenital deafness and Gilles de la Tourette Syndrome (GTS). GTS is characterised by multiple motor tics and one or more vocal tics that wax and wane. Due to his deafness Sam never developed vocal language but instead used sign language from the age of four. His tic disorder rapidly accelerated from the age of seven over a six-month period and soon sign language was incorporated into tics as complex "vocal" tics. Bursting out "words" in sign language would also occur in front of people unfamiliar with sign language and often with an obscene content although this was not evident to someone not trained in sign language. To our knowledge this is the first reported case of a congenital deaf child with GTS. The case presented here supports previously published work that the intentional share of the tics in GTS is very small. This case also questions former theories on which regions and circuits of the brain are involved in GTS.

Altered patterns of gene expression in response to myocardial infarction.

The use of cDNA microarrays has made it possible to simultaneously analyze gene expression for thousands of genes. Microarray technology was used to evaluate the expression of >4000 genes in a rat model of myocardial infarction. More than 200 genes were identified that showed differential expression in response to myocardial infarction. Gene expression changes were monitored from 2 to 16 weeks after infarction in 2 regions of the heart, the left ventricle free wall and interventricular septum. A novel clustering program was used to identify patterns of expression within this large set of data. Unique patterns were revealed within the transcriptional responses that illuminate changes in biological processes associated with myocardial infarction.