Transcriptomic analysis of Anopheles gambiae from Benin reveals overexpression of salivary and cuticular proteins associated with cross-resistance to pyrethroids and organophosphates.

BACKGROUND: Insecticide resistance (IR) is one of the major threats to malaria vector control programs in endemic countries. However, the mechanisms underlying IR are poorly understood. Thus, investigating gene expression patterns related to IR can offer important insights into the molecular basis of IR in mosquitoes. In this study, RNA-Seq was used to characterize gene expression in Anopheles gambiae surviving exposure to pyrethroids (deltamethrin, alphacypermethrin) and an organophosphate (pirimiphos-methyl). RESULTS: Larvae of An. gambiae s.s. collected from Bassila and Djougou in Benin were reared to adulthood and phenotyped for IR using a modified CDC intensity bottle bioassay. The results showed that mosquitoes from Djougou were more resistant to pyrethroids (5X deltamethrin: 51.7% mortality; 2X alphacypermethrin: 47.4%) than Bassila (1X deltamethrin: 70.7%; 1X alphacypermethrin: 77.7%), while the latter were more resistant to pirimiphos-methyl (1.5X: 48.3% in Bassila and 1X: 21.5% in Djougou). RNA-seq was then conducted on resistant mosquitoes, non-exposed mosquitoes from the same locations and the laboratory-susceptible An. gambiae s.s. Kisumu strain. The results showed overexpression of detoxification genes, including cytochrome P450s (CYP12F2, CYP12F3, CYP4H15, CYP4H17, CYP6Z3, CYP9K1, CYP4G16, and CYP4D17), carboxylesterase genes (COEJHE5E, COE22933) and glutathione S-transferases (GSTE2 and GSTMS3) in all three resistant mosquito groups analyzed. Genes encoding cuticular proteins (CPR130, CPR10, CPR15, CPR16, CPR127, CPAP3-C, CPAP3-B, and CPR76) were also overexpressed in all the resistant groups, indicating their potential role in cross resistance in An. gambiae. Salivary gland protein genes related to 'salivary cysteine-rich peptide' and 'salivary secreted mucin 3' were also over-expressed and shared across all resistant groups. CONCLUSION: Our results suggest that in addition to metabolic enzymes, cuticular and salivary gland proteins could play an important role in cross-resistance to multiple classes of insecticides in Benin. These genes warrant further investigation to validate their functional role in An. gambiae resistance to insecticides.

Adaptive modeling optimized by the data fusion strategy: Real-time dying cell percentage prediction using capacitance spectroscopy.

The previous research showcased a partial least squares (PLS) regression model accurately predicting cell death percentages using in-line capacitance spectra. The current study advances the model accuracy through adaptive modeling employing a data fusion approach. This strategy enhances prediction performance by incorporating variables from the Cole-Cole model, conductivity and its derivatives over time, and Mahalanobis distance into the predictor matrix (X-matrix). Firstly, the Cole-Cole model, a mechanistic model with parameters linked to early cell death onset, was integrated to enhance prediction performance. Secondly, the inclusion of conductivity and its derivatives over time in the X-matrix mitigated prediction fluctuations resulting from abrupt conductivity changes during process operations. Thirdly, Mahalanobis distance, depicting spectral changes relative to a reference spectrum from a previous time point, improved model adaptability to independent test sets, thereby enhancing performance. The final data fusion model substantially decreased root-mean squared error of prediction (RMSEP) by around 50%, which is a significant boost in prediction accuracy compared to the prior PLS model. Robustness against reference spectrum selection was confirmed by consistent performance across various time points. In conclusion, this study illustrates that the data fusion strategy substantially enhances the model accuracy compared to the previous model relying solely on capacitance spectra.

Capacitance spectroscopy enables real-time monitoring of early cell death in mammalian cell culture.

BACKGROUND/AIMS: Previous work developed a quantitative model using capacitance spectroscopy in an at-line setup to predict the dying cell percentage measured from a flow cytometer. This work aimed to transfer the at-line model to monitor lab-scale bioreactors in real-time, waiving the need for frequent sampling and enabling precise controls. METHODS AND RESULTS: Due to the difference between the at-line and in-line capacitance probes, direct application of the at-line model resulted in poor accuracy and high prediction bias. A new model with a variable range and offering similar spectral shape across all probes was first constructed, improving prediction accuracy. Moreover, the global calibration method included the variance of different probes and scales in the model, reducing prediction bias. External parameter orthogonalization, a preprocessing method, also mitigated the interference from feeding, which further improved model performance. The root-mean-square error of prediction of the final model was 6.56% (8.42% of the prediction range) with an R2 of 92.4%. CONCLUSION: The culture evolution trajectory predicted by the in-line model captured the cell death and alarmed cell death onset earlier than the trypan blue exclusion test. Additionally, the incorporation of at-line spectra following orthogonal design into the calibration set was shown to generate calibration models that are more robust than the calibration models constructed using the in-line spectra only. This is advantageous, as at-line spectral collection is easier, faster, and more material-sparing than in-line spectra collection.

Rapid at-line early cell death quantification using capacitance spectroscopy.

Cell death is one of the failure modes of mammalian cell culture. Apoptosis is a regulated cell death process mainly observed in cell culture. Timely detection of apoptosis onset allows opportunities for preventive controls that ensure high productivity and consistent product quality. Capacitance spectroscopy captures the apoptosis-related cellular properties changes and thus quantifies the percentage of dying cells. This study demonstrated a quantification model that measures the percentage of apoptotic cells using a capacitance spectrometer in an at-line setup. When predicting the independent test set collected from bench-scale bioreactors, the root-mean-squared error of prediction was 8.8% (equivalent to 9.9% of the prediction range). The predicted culture evolution trajectory aligned with measured values from the flow cytometer. Furthermore, this method alarms cell death onset earlier than the traditional viability test, that is, the trypan blue exclusion test. Compared to flow cytometry (the traditional early cell death detection method), this method is rapid, simple, and less labor-intensive. In addition, this at-line setup can be easily transferred between scales (e.g., lab-scale for development to manufacturing scale), which benefits process transfers between facilities, scale-up, and other process transitions.

First-Principles and Empirical Approaches to Predicting In Vitro Dissolution for Pharmaceutical Formulation and Process Development and for Product Release Testing.

This manuscript represents the perspective of the Dissolution Working Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) and of two focus groups of the American Association of Pharmaceutical Scientists (AAPS): Process Analytical Technology (PAT) and In Vitro Release and Dissolution Testing (IVRDT). The intent of this manuscript is to show recent progress in the field of in vitro predictive dissolution modeling and to provide recommended general approaches to developing in vitro predictive dissolution models for both early- and late-stage formulation/process development and batch release. Different modeling approaches should be used at different stages of drug development based on product and process understanding available at those stages. Two industry case studies of current approaches used for modeling tablet dissolution are presented. These include examples of predictive model use for product development within the space explored during formulation and process optimization, as well as of dissolution models as surrogate tests in a regulatory filing. A review of an industry example of developing a dissolution model for real-time release testing (RTRt) and of academic case studies of enabling dissolution RTRt by near-infrared spectroscopy (NIRS) is also provided. These demonstrate multiple approaches for developing data-rich empirical models in the context of science- and risk-based process development to predict in vitro dissolution. Recommendations of modeling best practices are made, focused primarily on immediate-release (IR) oral delivery products for new drug applications. A general roadmap is presented for implementation of dissolution modeling for enhanced product understanding, robust control strategy, batch release testing, and flexibility toward post-approval changes.

Breaking paradigms, new breast cancer rehabilitation methods from occupational therapy: case report / Rompiendo paradigmas: nuevas formas de rehabilitación para el cáncer de mama desde terapia ocupacional. Reporte de caso

Introduction: Breast cancer is one of the most frequent diseases in Colombia and worldwide. Thousands of women, who undergo treatment, survive and require timely and comprehensive occupational therapy intervention. This paper presents a rehabilitation case study that followed the biopsychosocial and quality of life in persons model. Case presentation: Intervention on a 64-year-old woman referred to the occupational therapy service with a diagnosis of infiltrating ductal carcinoma of the left breast with neoadjuvant radiotherapy, after modified radical mastectomy and stage III lymphedema. She presented with restricted participation and occupational performance, specifically in activities of daily living, with relevant psychosocial and socio-emotional consequences. An intervention focused on the individual, following a biopsychosocial approach, was proposed in order to apply strategies on restorative, empowerment and maintenance activities of occupational skills involved in activities of daily living. Emphasis was placed on socio-emotional, occupational biomechanics and education aspects with special care to involve the interests of women. Conclusions: Rehabilitation for breast cancer patients not only involves biomedical care but also approaches psychosocial aspects that sometimes have to be solved in advance to get results that are later evident in the health of the person. In this case, a breast prosthesis was elaborated by and for the person, using all kinds of strategies that responded to biomedical axes and well-being and health

Introducción. El cáncer de mama es una enfermedad recurrente en el mundo y en Colombia. Miles de mujeres que la padecen se someten a tratamiento, sobreviven y necesitan una oportuna, y sobre todo holística, intervención desde la terapia ocupacional. Se presenta un estudio de caso de rehabilitación en concordancia al modelo biopsicosocial y centrado en la persona. Presentación del caso. Mujer de 64 años remitida al servicio de terapia ocupacional con diagnóstico de cáncer ductal infiltrante de mama izquierda con neoadyuvancia por radioterapia, posterior a mastectomía radical modificada y linfedema etapa III, quien presenta restricciones en la participación y desempeño ocupacional, en específico en actividades de la vida diaria con alteraciones psicosociales y socioemocionales relevantes. Se planteó una intervención centrada en la persona, con enfoque biopsicosocial, en donde se aplicaron diferentes estrategias en actividades de tipo restaurativo, potenciación y mantenimiento de destrezas ocupacionales implicadas en actividades de la vida diaria. Se hizo énfasis en las áreas socioemocional, de biomecánica ocupacional y de educación con especial cuidado de involucrar los intereses de la mujer. Conclusiones. La rehabilitación del cáncer de mama no solo implica atención biomédica, sino también abordaje de aspectos psicosociales que en ocasiones tienen que ser resueltos con antelación para conseguir resultados que se evidencien en la salud. En este caso la elaboración de una prótesis de mama por y para la persona vinculó toda clase de estrategias que respondían a ejes biomédicos y comprendían en su totalidad el bienestar y la salud

Infusion of freshly isolated autologous bone marrow derived mononuclear cells prevents endotoxin-induced lung injury in an ex-vivo perfused swine model.

INTRODUCTION: The acute respiratory distress syndrome (ARDS), affects up to 150,000 patients per year in the United States. We and other groups have demonstrated that bone marrow derived mesenchymal stromal stem cells prevent ARDS induced by systemic and local administration of endotoxin (lipopolysaccharide (LPS)) in mice. METHODS: A study was undertaken to determine the effects of the diverse populations of bone marrow derived cells on the pathophysiology of ARDS, using a unique ex-vivo swine preparation, in which only the ventilated lung and the liver are perfused with autologous blood. Six experimental groups were designated as: 1) endotoxin alone, 2) endotoxin + total fresh whole bone marrow nuclear cells (BMC), 3) endotoxin + non-hematopoietic bone marrow cells (CD45 neg), 4) endotoxin + hematopoietic bone marrow cells (CD45 positive), 5) endotoxin + buffy coat and 6) endotoxin + in vitro expanded swine CD45 negative adherent allogeneic bone marrow cells (cultured CD45neg). We measured at different levels the biological consequences of the infusion of the different subsets of cells. The measured parameters were: pulmonary vascular resistance (PVR), gas exchange (PO2), lung edema (lung wet/dry weight), gene expression and serum concentrations of the pro-inflammatory cytokines IL-1ß, TNF-α and IL-6. RESULTS: Infusion of freshly purified autologous total BMCs, as well as non-hematopoietic CD45(-) bone marrow cells significantly reduced endotoxin-induced pulmonary hypertension and hypoxemia and reduced the lung edema. Also, in the groups that received BMCs and cultured CD45neg we observed a decrease in the levels of IL-1ß and TNF-α in plasma. Infusion of hematopoietic CD45(+) bone marrow cells or peripheral blood buffy coat cells did not protect against LPS-induced lung injury. CONCLUSIONS: We conclude that infusion of freshly isolated autologous whole bone marrow cells and the subset of non-hematopoietic cells can suppress the acute humoral and physiologic responses induced by endotoxemia by modulating the inflammatory response, mechanisms that do not involve engraftment or trans-differentiation of the cells. These observations may have important implications for the design of future cell therapies for ARDS.

Comparison of near infrared and microwave resonance sensors for at-line moisture determination in powders and tablets.

In this paper we demonstrate the feasibility of replacing KF for water content testing in bulk powders and tablets with at-line near infrared (NIR) or microwave resonance (MR) methods. Accurate NIR and MR prediction models were developed with a minimalistic approach to calibration. The NIR method can accurately predict water content in bulk powders in the range of 0.5-5% w/w. Results from this method were compared to a MR method. We demonstrated excellent agreement of both NIR and MR methods for powders vs. the reference KF method. These methods are applicable to in-process control or quality control environments. One of the aims of this study was to determine if a calibration developed for a particular product could be used to predict the water content of another product (with related composition) but containing a different active pharmaceutical ingredient (API). We demonstrated that, contrary to the NIR method, a general MR method can be used to predict water content in two different types of blends. Finally, we demonstrated that a MR method can be developed for at-line moisture determination in tablets.

Drug interactions with potential rubber closure extractables. Identification of thiol-disulfide exchange reaction products of captopril and thiurams.

Mixtures of thiuram disulfides are frequently used as accelerators in rubber stoppers for injectables and sterilized powders for injection. Rapid reactions of thiuram disulfides between themselves and with thiols yield mixed disulfides due to thiol-disulfide exchange. The possibility of exchange reactions of thiuram disulfides extracted from rubber stoppers and drug products containing pendant thiol groups have not been reported in the analysis of potential stopper extractables. In this paper we report the formation and identification of mixed thiuram disulfides of N,N,N',N'-dimethylthiuram disulfide (TMTD), N,N,N',N'-dibutylthiuram disulfide (TBTD), and captopril (a thiol-containing drug). A reversed-phase HPLC method was developed for the determination of TMTD, TBTD, captopril and their disulfides in aqueous vehicles, using a YMC ODS AQ column at 35 degrees C and mobile phases A and B consisting of acetonitrile:water:trifluoroacetic acid (TFA) (20:80:0.1) and acetonitrile:TFA (100:0.1), respectively. The captopril-TBTD and captopril-TMTD disulfides were identified by MS, with molecular ions at m/z 420.9 and m/z of 337.1, respectively. Possible structures for the fragment ions in the spectra are provided. Mixed captopril-thiuram formation was studied as a function of pH. Captopril-TMTD formation was enhanced at pH 6.0, reaching a maximum of 31.3% in 4.1h. At pH 4.0 and 2.2, the mixed captopril adduct product was still detected in solution after 20h. The impact of the formation of mixed disulfide products of thiol-containing drugs with thiurams in the HPLC profile of extractables and leachables studies is discussed.

Comprehensive determination of extractables from five different brands of stoppers used for injectable products.

Five commonly used stopper formulations were tested for extractables using three different vehicles (pH 3 citrate buffer with 20% w/v sulfobutylether-beta-cyclodextrin, pH 8 phosphate buffer and 50/50 v/v polyoxyethylated castor oil/dehydrated alcohol). The stoppers, made from butyl and halobutyl rubbers, coated and uncoated with proprietary films, were stored in contact with each vehicle for up to 6 months at 40 degrees C/75% relative humidity (RH) or for up to 24 months at 25 degrees C/60% RH. Samples were analyzed for the presence of extractables using inductively coupled plasma-atomic emission spectroscopy, ion chromatography, high-performance liquid chromatography, and gas chromatography. Extractables were observed at greater than 10 ppm for only one of the five stoppers that were tested. Based on these results, a standardized protocol for stopper extractable testing was developed. This protocol has been used to satisfy stopper extractable testing regulatory requirements for a number of different new injectable products.

Two-photon photochromism of a diarylethene enhanced by Förster resonance energy transfer from two-photon absorbing fluorenes.

Resonance energy transfer from two-photon absorbing fluorene derivatives to the photochromic compound 3,4-bis-(2,4,5-trimethyl-thiophen-3-yl)furan-2,5-dione (PC 1) is investigated in hexane under one- and two-photon excitation. The quenching of the steady-state fluorescence of donor molecules in the presence of the diarylethene acceptor is used to study the nature of resonance energy transfer. The Förster distances and critical acceptor concentrations are determined for nonbound donor-acceptor pairs in homogeneous molecular ensembles. Quite significantly, up to a two-fold enhancement in the velocity of the photochromic transformation of 1, in the presence of two-photon absorbing fluorene derivatives, is demonstrated.

Activation of alveolar macrophages via the alternative pathway in herpesvirus-induced lung fibrosis.

The etiology of idiopathic pulmonary fibrosis (IPF) is unknown. Because viral pathogenesis of IPF has been suggested, we have established a murine model of progressive pulmonary fibrosis by infecting IFN-gammaR-deficient mice (IFN-gammaR(-/-)) with the murine gamma-herpesvirus 68. Because alveolar macrophages in humans with IPF have been implicated in driving the profibrotic response, we studied their role in our model. Chronic herpesvirus infection of the lung was associated with recruitment of alveolar macrophages to areas with epithelial hyperplasia and fibrosis in infected lungs. Using immunohistochemistry, Western blot, and RT-PCR techniques, we demonstrated that recruited alveolar macrophages showed high levels of expression of the proteins Ym1/2, FIZZ1 (found in inflammatory zone 1), insulin-like growth factor-1, and arginase I, and also active transcription of fibronectin, indicative of activation of macrophages by an alternative pathway. Arginase I expression was also evident in interstitial fibroblasts, and increased arginase activity was found in lungs of infected animals. Lung tissue from patients with IPF showed increased expression of arginase I in epithelial cells, fibroblast foci, and alveolar macrophages compared with normal lung. These results suggest that virus-induced upregulation of arginase I could be a mechanism driving lung fibrogenesis.

Synthesis and characterization of new fluorene-based singlet oxygen sensitizers.

The synthesis, photophysical characterization, and determination of singlet oxygen quantum yields (Phi(Delta)) for a class of fluorene derivatives with potential application in two-photon photodynamic therapy (PDT) is reported. It has been demonstrated that these compounds possess the ability to generate singlet oxygen (1O2) upon excitation. A photochemical method, using 1,3-diphenylisobenzofuran (DPBF) as 1O2 chemical quencher, was employed to determine the singlet oxygen quantum yields (Phi(Delta)) of the fluorene-based photosensitizers in ethanol. Phi(Delta) values ranged from 0.35 to 0.75. These derivatives may have potential application as two-photon photosensitizers when pumped via two-photon excitation in the near-IR spectral region.

Sarcoma de kaposi del tracto gastrointestinal como primera manifestación de SIDA

Ilustramos el caso de un paciente de 40 años de edad, sexo masculino, homosexual, con sarcoma de kaposi gastrointestinal sin lesiones cutáneas como primera manifestación de SIDA, quien presentaba lesión en el dorso de la lengua de aspecto polipoide y violácea asociada a pólipos rectales. Se realizaron estudios de vías digestivas altas y colon enema de doble contraste los cuales evidenciaron lesiones nodulares son umbilicación central y compresiones extrínsecas en la mayor parte del tracto gastrointestinal. Se confirmó diagnostico de sarcoma de kaposi mediante biopsia de las lesiones.El reporte de este caso único quiere ilustrar la presentación radiológica del sarcoma de Kaposi en el tracto gastrointestinal sin compromiso cutáneo y como primera manifestación de SIDA.

Quiste infectado del uraco en un niño con dolor abdominal agudo

Presentación de un caso de quiste del uraco infectado, y discusión de su apariencia imaginológica

Desarrollo de un modelo cuantitativo estructura-actividad (QSAR) basado en índices de conectividad molecular, para antihelmínticos del tipo benzimidazol / Development of a quantitative structured-activity (QSAR) based model in index of molecular connectivity, for anthelmintic of benzimidazol type

Se estableció una relación cuantitativa entre la actividad antihelmíntica de un grupo de benzimidazoles 2-metil carbamato 5(6) sustituidos (expresada como IC50 o concentración micromolar requerida para inhibir el 50 por ciento de la polimerización de la tubulina) y su estructura química, la cual fué cuantificada por medio de indices de Chi(X) conectividad molecular, propuestos por Kier y Hall. Para relacionar los valores experimentales de actividad antihelmíntica (IC50) y los indices de conectividad molecular calculados (ºX,ºXv,1X, 1Xv, 3Xp, 3Xpv, 3Xc, 3Xvc, 4Xp, 4XpV, 4Xpcv, 6Xp, 6Xpv,6Xpc 6Xpcv) se empleó análisis estadístico de regresión lineal simple y múltiple, con base en el cual se escogió el mejor modelo QSAR. Se observó que la actividad antihelmíntica de 31 benzimidazoles, se relaciona linealmente con los índices de conectividad molecular del tipo 4Xpc, 6Xpcv, 6Xp, lo cual explica la dependencia de la actividad con la longitud, el contenido de heteroátomos y la ramificación del sustituyente en la posición 5 del anillo benzimidazol. El modelo propuesto permitió predecir la actividad de otros benzimidazoles, estructuralmente relacionados con los compuestos en estudio. 6 nuevas moléculas, de un grupo nueve analizadas, presentan buena actividad de acuerdo a este modelo

Utilidad de la medición de niveles séricos de anticuerpos específicos anti E. Histolitica como diagnóstico de amibiasis invasiva V. Y. G. absceso hepático amebiano, en la población general / Usefulness of measurement of seric level of specific anti-E. histolityca antibodies as diagnosis of invasive amebiasis v., and g. amebic liver abscess in general population

El presente trabajo es el desarrollo de un marco teórico sobre los conocimientos actuales de amibiasis, haciendo énfasis en los conceptos inmunológicos, para poder realizar un trabajo práctico eque establezca la utilidad de las pruebas serológicas en el diagnóstico de las formas extraintestinales invasivas como el absceso hepático amebiano. Los resultados de la investigación se obtuvieron gracias a la asesoria en inmunología y pruebas de laboratorio (serología), en conjunto con la revisión bibliográfica amplia de estudios realizados en México, USA e India. Se estableció que método ELISA y wk IHA, son los más confiables por su sensibilidad y especificidad. Dado que Colombia posee una de las tasas mas altas de incidencia de amibiasis en el mundo y la gran utilidad de las pruebas serologicas, es necesario estandarizarlas en nuestro medio, aunque somos concients de que los costos son un obstáculo y el espectro clinico de la enfermedad es multifactorial