Measuring cfDNA integrity as a biomarker for predicting neoadjuvant chemotherapy response in breast cancer patients: a pilot study.

PURPOSE: Circulating cell-free DNA (cfDNA) is a promising biomarker for predicting treatment response and disease outcomes in Breast Cancer (BC) patients undergoing neoadjuvant chemotherapy (NAC). To determine if cfDNA originates from tumors, matching tumor and cfDNA gene mutations are necessary, often requiring tumor DNA sequencing. We assessed plasma cfDNA integrity by measuring concentrations and ratios of larger-to-smaller Alu DNA fractions as a potential biomarker, eliminating the need for prior tumor sequencing. METHODS: We included patients with localized and/or locally advanced BC receiving standard NAC alone or in combination with immunotherapy and/or anti-HER2 targeted therapy. Blood samples were collected before treatment, every 2 weeks during treatment, and before surgery. RESULTS: Of the 38 evaluated patients, only 28 completed the protocol and underwent surgery after NAC. Seven patients (25%) achieved a pathologic complete response (pCR). We found that cfDNA integrity (cfDNAI) levels at 15 days after starting NAC were significantly higher in patients who achieved pCR (p = 0.045) and correlated significantly with Disease-Free Survival (DFS) in univariate analysis (p = 0.0371). CONCLUSIONS: Evaluation of cfDNAI 2 weeks after NAC initiation appears to be an early biomarker for tumor pCR and DFS. Measuring Alu fragments of different lengths may replace techniques requiring prior tumor sequencing to measure ctDNA, reducing costs and complexity of cfDNA serial measurements in BC patients undergoing NAC.

Deconstructing the long-standing a priori assumption that serial homology generally involves ancestral similarity followed by anatomical divergence.

It has long been assumed that serial homologues are ancestrally similar-polysomerism resulting from a "duplication" or "repetition" of forms-and then often diverge-anisomerism, for example, as they become adapted to perform different tasks as is the case with the forelimb and hind limbs of humans. However, such an assumption, with crucial implications for comparative, evolutionary, and developmental biology, and for evolutionary developmental biology, has in general not really been tested by a broad analysis of the available empirical data. Perhaps not surprisingly, more recent anatomical comparisons, as well as molecular knowledge of how, for example, serial appendicular structures are patterned along with different anteroposterior regions of the body axis of bilateral animals, and how "homologous" patterning domains do not necessarily mark "homologous" morphological domains, are putting in question this paradigm. In fact, apart from showing that many so-called "serial homologues" might not be similar at all, recent works have shown that in at least some cases some "serial" structures are indeed more similar to each other in derived taxa than in phylogenetically more ancestral ones, as pointed out by authors such as Owen. In this article, we are taking a step back to question whether such assumptions are actually correct at all, in the first place. In particular, we review other cases of so-called "serial homologues" such as insect wings, arthropod walking appendages, Dipteran thoracic bristles, and the vertebrae, ribs, teeth, myomeres, feathers, and hairs of chordate animals. We show that: (a) there are almost never cases of true ancestral similarity; (b) in evolution, such structures-for example, vertebra-and/or their subparts-for example, "transverse processes"-many times display trends toward less similarity while in many others display trends toward more similarity, that is, one cannot say that there is a clear, overall trend to anisomerism.

First use of anatomical networks to study modularity and integration of heads, forelimbs and hindlimbs in abnormal anencephalic and cyclopic vs normal human development.

The ill-named "logic of monsters" hypothesis of Pere Alberch - one of the founders of modern evo-devo - emphasized the importance of "internal rules" due to strong developmental constraints, linked teratologies to developmental processes and patterns, and contradicted hypotheses arguing that birth defects are related to a chaotic and random disarray of developmental mechanisms. We test these hypotheses using, for the first time, anatomical network analysis (AnNA) to study and compare the musculoskeletal modularity and integration of both the heads and the fore- and hindlimbs of abnormal cyclopic trisomy 18 and anencephalic human fetuses, and of normal fetal, newborn, and adult humans. Our previous works have shown that superficial gross anatomical analyses of these specimens strongly support the "logic of monsters" hypothesis, in the sense that there is an 'order' or 'logic' within the gross anatomical patterns observed in both the normal and abnormal individuals. Interestingly, the results of the AnNA done in the present work reveal a somewhat different pattern: at least concerning the musculoskeletal modules obtained in our AnNA, we observe a hybrid between the "logic of monsters" and the "lack of homeostasis" hypotheses. For instance, as predicted by the latter hypothesis, we found a high level of left-right asymmetry in the forelimbs and/or hindlimbs of the abnormal cyclopic trisomy 18 and anencephalic human fetuses. That is, a network analysis of the organization of/connection between the musculoskeletal structures of these fetuses reveals a more "chaotic" pattern than that detected by superficial gross anatomical comparisons. We discuss the broader developmental, evolutionary, and medical implications of these results.

Musculoskeletal study of cebocephalic and cyclopic lamb heads illuminates links between normal and abnormal development, evolution and human pathologies.

This paper is part of the emerging field of Evolutionary Developmental Pathology, dedicated to study the links between normal and abnormal development, evolution and human pathologies. We analyzed the head musculoskeletal system of several 'natural mutant' newborn lambs displaying various degrees of abnormality, from mild defects to cebocephaly and to cyclopia, and compared them with humans. Interestingly, muscle defects are less marked than osteological ones, and contrarily to the latter they tend to display left-right assymetries. In individuals with cebocephalic and even cyclopic skulls almost all head muscles are normal. The very few exceptions are some extraocular muscles and facial muscles that normally attach to osteological structures that are missing in the abnormal heads: such muscles are instead attached to the 'nearest topological neighbor' of the missing osteological structure, a pattern also found in cyclopic humans. These observations support Alberch's ill-named "logic of monsters" - as a byproduct of strong developmental/topological constraints anatomical patterns tend to repeat themselves, even severe malformations displayed by distantly related taxa. They also support the idea that mammalian facial muscles reverted to an ancestral 'nearest-neighbor' muscle-bone type of attachment seen in non-vertebrate animals and in vertebrate limbs, but not in other vertebrate head muscles.

Transgenic Nicotiana tabacum seeds expressing the Mycobacterium tuberculosis Alanine- and Proline-rich antigen.

The glycoprotein APA (Alanine- and Proline-rich Antigen, a 45/47 kDa antigen complex, Rv1860) is considered as a major immunodominant antigen secreted by M. tuberculosis. This antigen has proved to be highly immunogenic in experimental models and humans, presenting a significant potential for further development of a new vaccine for tuberculosis. Glycosylation plays a key role in the immunogenicity of the APA protein. Because plants are known to promote post-translational modification such as glycosylation and to be one of the most economic and safe hosts for recombinant protein expression, we have over expressed the APA protein in transgenic tobacco plants aiming to produce a glycosylated version of the protein. Seeds are known to be a well-suited organ to accumulate recombinant proteins, due to low protease activity and higher protein stability. We used a seed-specific promoter from sorghum, a signal peptide to target the protein to the endoplasmic reticulum and ultimately in the protein storage vacuoles. We show that the recombinant protein accumulated in the seeds had similar isoelectric point and molecular weight compared with the native protein. These findings demonstrate the ability of tobacco plants to produce glycosylated APA protein, opening the way for the development of secure, effective and versatile vaccines or therapeutic proteins against tuberculosis.

Transgenic Nicotiana tabacum seeds expressing the Mycobacterium tuberculosis Alanine- and Proline-rich antigen

The glycoprotein APA (Alanine- and Proline-rich Antigen, a 45/47 kDa antigen complex, Rv1860) is considered as a major immunodominant antigen secreted by M. tuberculosis. This antigen has proved to be highly immunogenic in experimental models and humans, presenting a significant potential for further development of a new vaccine for tuberculosis. Glycosylation plays a key role in the immunogenicity of the APA protein. Because plants are known to promote post-translational modification such as glycosylation and to be one of the most economic and safe hosts for recombinant protein expression, we have over expressed the APA protein in transgenic tobacco plants aiming to produce a glycosylated version of the protein. Seeds are known to be a well-suited organ to accumulate recombinant proteins, due to low protease activity and higher protein stability. We used a seed-specific promoter from sorghum, a signal peptide to target the protein to the endoplasmic reticulum and ultimately in the protein storage vacuoles. We show that the recombinant protein accumulated in the seeds had similar isoelectric point and molecular weight compared with the native protein. These findings demonstrate the ability of tobacco plants to produce glycosylated APA protein, opening the way for the development of secure, effective and versatile vaccines or therapeutic proteins against tuberculosis.

Transgenic Nicotiana tabacum seeds expressing the Mycobacterium tuberculosis Alanine- and Proline-rich antigen

The glycoprotein APA (Alanine- and Proline-rich Antigen, a 45/47 kDa antigen complex, Rv1860) is considered as a major immunodominant antigen secreted by M. tuberculosis. This antigen has proved to be highly immunogenic in experimental models and humans, presenting a significant potential for further development of a new vaccine for tuberculosis. Glycosylation plays a key role in the immunogenicity of the APA protein. Because plants are known to promote post-translational modification such as glycosylation and to be one of the most economic and safe hosts for recombinant protein expression, we have over expressed the APA protein in transgenic tobacco plants aiming to produce a glycosylated version of the protein. Seeds are known to be a well-suited organ to accumulate recombinant proteins, due to low protease activity and higher protein stability. We used a seed-specific promoter from sorghum, a signal peptide to target the protein to the endoplasmic reticulum and ultimately in the protein storage vacuoles. We show that the recombinant protein accumulated in the seeds had similar isoelectric point and molecular weight compared with the native protein. These findings demonstrate the ability of tobacco plants to produce glycosylated APA protein, opening the way for the development of secure, effective and versatile vaccines or therapeutic proteins against tuberculosis.

Otologic and audiologic outcomes with the Furlow and von Langenbeck with intravelar veloplasty palatoplasties in unilateral cleft lip and palate.

OBJECTIVE: Cleft palate increases the risk of chronic middle ear disease and hearing loss. The goal of this report was to determine which of two palate surgeries and which timing of palate surgery were associated with better otologic and audiologic outcomes in children with unilateral cleft lip and palate at 5 to 6 years of age. DESIGN: Subjects were randomly assigned to the von Langenbeck with intravelar veloplasty or Furlow palate repair, to palate surgery at 9 to 12 months or 15 to 18 months of age, and to the Spina or Millard lip repair. SETTING: Centralized, tertiary care craniofacial treatment center. PATIENTS: A total of 673 infants with unilateral cleft lip and palate. INTERVENTIONS: Palate and lip were repaired using established techniques. Serial otoscopic and audiometric evaluations were performed. MAIN OUTCOME MEASURES: Hearing and otoscopic findings at 5 to 6 years old. RESULTS: There were 370 children available for analysis. Hearing and need for tympanostomy tube placement did not differ by palatoplasty, age at palatoplasty, cheiloplasty, or surgeon. Risk of developing cholesteatoma or perforation was higher with Millard cheiloplasty (odds ratio  =  5.1, 95% confidence interval  =  1.44 to 18.11, p  =  .012). Type and age at palatoplasty were not significantly associated with either the rate of developing these sequelae or the rate of achieving bilaterally normal hearing and ear examinations. CONCLUSIONS: Type of palatoplasty did not influence otologic and audiologic outcomes in 5- to 6-year-olds with unilateral cleft lip and palate. The potential influence of lip repair on otologic outcomes warrants further investigation.

Longitudinal study of growth of children with unilateral cleft-lip palate from birth to two years of age.

OBJECTIVE: To study the growth of children with complete unilateral cleft lip and palate (UCLP) from birth to 2 years of age and to construct specific UCLP growth curves. DESIGN: Physical growth was a secondary outcome measure of a National Institutes of Health-sponsored longitudinal, prospective clinical trial involving the University of Florida (United States) and the University of São Paulo (Brazil). PATIENTS: Six hundred twenty-seven children with UCLP, nonsyndromic, both genders. METHODS: Length, weight, and head circumference were prospectively measured for a group of children enrolled in a clinical trial. Median growth curves for the three parameters (length, weight, head circumference) were performed and compared with the median for the National Center for Health Statistics (NCHS) curves. The median values for length, weight, and head circumference at birth and 6, 12, 18, and 24 months of age were plotted against NCHS median values and statistically compared at birth and 24 months. SETTING: Hospital de Reabilitação de Anomalias Craniofaciais, Universidade de São Paulo, Bauru, Brazil (HRAC-USP). RESULTS: At birth, children of both genders with UCLP presented with smaller body dimensions in relation to NCHS median values, but the results suggest a catch-up growth for length, weight, and head circumference for girls and for weight (to some degree) and head circumference for boys. CONCLUSIONS: Weight was the most compromised parameter for both genders, followed by length and then head circumference. There was no evidence of short stature. This study established growth curves for children with UCLP.

An enzyme-linked immunosorbent assay for the detection of human seminal plasma inhibin.

A simple and sensitive enzyme-linked immunosorbent assay (ELISA) is described for the measurement of inhibin in urine and seminal plasma. Standards used cover a range from 50 ng to 0.05 ng/0.1 ml with a detection limit of 0.098 ng/0.1 ml. Coefficients of variation for intra-assay precision and for inter-assay precision were obtained and compared favourably with RIA. Given the ease of application, this technique is an useful alternative to existing radioimmuno-assays for this peptide.