Intra-organ cell specific mitochondrial quantitative interactomics.

Almost every organ consists of many cell types, each with its unique functions. Proteomes of these cell types are thus unique too. But it is reasonable to assume that interactome (inter and intra molecular interactions of proteins) are also distinct since protein interactions are what ultimately carry out the function. Podocytes and tubules are two cell types within kidney with vastly different functions: podocytes envelop the blood vessels in the glomerulus and act as filters while tubules are located downstream of the glomerulus and are responsible for reabsorption of important nutrients. It has been long known that for tubules mitochondria plays an important role as they require a lot of energy to carry out their functions. In podocytes, however, it has been assumed that mitochondria might not matter as much in both normal physiology and pathology1. Here we have applied quantitative cross-linking mass spectrometry to compare mitochondrial interactomes of tubules and podocytes using a transgenic mitochondrial tagging strategy to immunoprecipitate cell-specific mitochondria directly from whole kidney. We have uncovered that mitochondrial proteomes of these cell types are quite similar, although still showing unique features that correspond to known functions, such as high energy production through TCA cycle in tubules and requirements for detoxification in podocytes which are on the frontline of filtration where they encounter toxic compounds and therefore, as a non-renewing cell type they must have ways to protect themselves from cellular toxicity. But we gained much deeper insight with the interactomics data. We were able to find pathways differentially regulated in podocytes and tubules based on changing cross-link levels and not just protein levels. Among these pathways are betaine metabolism, lysine degradation, and many others. We have also demonstrated how quantitative interactomics could be used to detect different activity levels of an enzyme even when protein abundances of it are the same between cell types. We have validated this finding with an orthogonal activity assay. Overall, this work presents a new view of mitochondrial biology for two important, but functionally distinct, cell types within the mouse kidney showing both similarities and unique features. This data can continue to be explored to find new aspects of mitochondrial biology, especially in podocytes, where mitochondria has been understudied. In the future this methodology can also be applied to other organs to uncover differences in the function of cell types.

Advocacy training during pediatric residency.

Despite broad concerns about the welfare of children, most pediatric residents are not able to engage in child advocacy during their busy training years. Yet residency can provide an opportunity for young pediatricians to learn valuable advocacy skills by undertaking an independent project with an experienced mentor. We describe the University of Washington Pediatrics Residency Program's experience in training interested residents in child advocacy. Basic requirements are that advocacy projects must not interfere with clinical training, resident participation must be voluntary, and faculty with advocacy skills must be available to help guide the residents. Four resident projects are outlined and guidelines for instituting such programs are presented.

A report of congenital indifference to pain as diagnosed in infancy.

As physicians and nurses, we have a fundamental obligation to consider patient welfare above all else. The case presented demonstrates the depth of this obligation. Our patient, who was diagnosed during infancy with congenital indifference to pain, can neither feel nor perceive the meaning of pain. Congenital indifference to pain is a rare disorder, which is characterized by normal intelligence, development, and sensation despite an absence of pain perception. There is no cure and treatment difficulties are plentiful. Educating and maintaining an open, consistent relationship with the family and caregivers is essential to reduce the morbidity of this condition.

Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations.

Congenital indifference to pain (CIP) is a rare condition in which patients have severely impaired pain perception, but are otherwise essentially normal. We identified and collected DNA from individuals from nine families of seven different nationalities in which the affected individuals meet the diagnostic criteria for CIP. Using homozygosity mapping and haplotype sharing methods, we narrowed the CIP locus to chromosome 2q24-q31, a region known to contain a cluster of voltage-gated sodium channel genes. From these prioritized candidate sodium channels, we identified 10 mutations in the SCN9A gene encoding the sodium channel protein Nav1.7. The mutations completely co-segregated with the disease phenotype, and nine of these SCN9A mutations resulted in truncation and loss-of-function of the Nav1.7 channel. These genetic data further support the evidence that Nav1.7 plays an essential role in mediating pain in humans, and that SCN9A mutations identified in multiple different populations underlie CIP.

Reduced growth velocity in exclusively breast-fed infants.

A number of recent studies have reported reduced growth velocity among breast-fed infants, as compared with standard growth curves. Contradictions between these and previous studies of breast-feeding have been difficult to resolve because of methodological problems, particularly supplementation of breast-feeding with other nutrients. In the present study, 33 term infants, exclusively breast-fed for six months, showed significantly slower rates of growth compared with data from the National Center for Health Statistics (NCHS). Between birth and 6 months of age, these infants lost an average of 20 percentiles in weight for age and 30 percentiles in length in relation to the NCHS population. We discuss these findings in terms of the appropriateness of the NCHS data as standards and the adequacy of exclusive breast-feeding for providing optimum growth through 6 months of age.

Clinical prediction of cervical spine injuries in children. Radiographic abnormalities.

During the seven-year period from 1976 through 1982, 2133 cervical spine radiographs were obtained for children less than 18 years of age at two hospitals in Tucson. Twenty-five children (1.2%) had x-ray evidence of a cervical spine injury, with a male to female ratio of 4:1. Vehicular accidents accounted for 36% of cervical spine radiographic abnormalities, and sports or playground accidents accounted for an additional 36%. In an attempt to find clinical predictors that might identify x-ray abnormalities, we reviewed the medical records of these 25 children with abnormalities and 713 randomly selected children without x-ray evidence of cervical spine injuries for the following: method of injury, presenting complaints, physical examination findings, therapy, and complications. No single clinical predictor had a sensitivity of 100% when considered in isolation, but clinical assessment consisting of EITHER a complaint of neck pain OR involvement in a vehicular accident with head trauma would have correctly identified all 25 cases of cervical spine injury. If this information had been used prospectively, the number of cervical spine radiographs ordered would have been reduced by 32%. We conclude that the use of this clinical "marker" would have positively identified all children with cervical spine injuries and would have reduced by one third the cost and radiation exposure associated with cervical spine radiographs. Because of the serious consequences of missing a cervical spine injury, we suggest that other studies confirm these results before this information is accepted as a recommendation.

[Risk factors for acute infantile diarrhea in a rural community in Chiapas, Mexico. A strategy for intervention]. / Factores de riesgo de diarrea infantil aguda en una communidad rural de Chiapas, México. Una estrategia de intervención.

A community-based, case-control study was conducted during the summer peak season for diarrhea in the highlands of Chiapas, Mexico, to identify risk and protective factors associated with acute diarrhea in children less than 6 years of age. To estimate the diarrheal morbidity rate, the community was divided into 13 sectors, each of about 20 households. A resident (volunteer mother) made daily visits to every household in her sector to identify new cases of diarrhea. During 3 weeks of surveillance, 63 children with diarrhea and 48 control children were identified. The diarrheal attack rate during this period for children less than 6 years of age was 30%. Analysis of 29 neighborhood-matched case-control pairs showed that children with diarrhea were more likely than their controls to have had a mother with diarrhea in the 2 weeks preceding the onset of the child's diarrhea (P less than 0.05; relative risk = 10). The association of childhood diarrhea with maternal diarrhea may serve as a focus for more detailed studies as well as an intervention that may be appropriate and effective for this community.

Risk factors for acute childhood diarrhea in the highlands of Chiapas, Mexico: a strategy for intervention.

A case-control study was conducted in the highlands of Chiapas, Mexico, to identify factors associated with acute diarrhea in children less than six years old. The study found that the diarrhea attack rate among the children surveyed during three weeks in the month of August (the peak diarrhea season) was approximately 30%, and that children whose mothers had diarrhea were especially likely to contract the illness themselves. The methods employed could prove relevant to studies in other areas, and the results obtained could provide the basis for more detailed study of the area involved--and for preventive action.

Purification and characterization of other distinct bone-inducing factors.

We purified a factor that induces bone formation greater than 300,000-fold from guanidinium chloride extracts of demineralized bone. Fifty nanograms of highly purified protein was active in an in vivo cartilage and bone-formation assay. The activity resided in a single gel band, corresponding to a molecular mass of approximately 30 kDa, which yielded proteins of 30, 18, and 16 kDa on reduction. The partial amino acid sequence obtained from these proteins confirmed our identification of specific factors that induce new bone formation in vivo.

Molecular cloning of a cDNA encoding interleukin 11, a stromal cell-derived lymphopoietic and hematopoietic cytokine.

Hematopoiesis occurs in close association with a complex network of cells loosely termed the hematopoietic microenvironment. Analysis of the mechanisms of microenvironmental regulation of hematopoiesis has been hindered by the complexity of the microenvironment as well as the heterogeneity of hematopoietic stem cells and early progenitor cells. We have established immortalized primate bone marrow-derived stromal cell lines to facilitate analysis of the interactions of hematopoietic cells with the microenvironment in a large animal species. One such line, PU-34, was found to produce a variety of growth factors, including an activity that stimulates the proliferation of an interleukin 6-dependent murine plasmacytoma cell line. A cDNA encoding the plasmacytoma stimulatory activity was isolated through functional expression cloning in mammalian cells. The nucleotide sequence contained a single long reading frame of 597 nucleotides encoding a predicted 199-amino acid polypeptide. The amino acid sequence of this cytokine, designated interleukin 11 (IL-11), did not display significant similarity with any other sequence in the GenBank data base. Preliminary biological characterization indicates that in addition to stimulating plasmacytoma proliferation, IL-11 stimulates the T-cell-dependent development of immunoglobulin-producing B cells and synergizes with IL-3 in supporting murine megakaryocyte colony formation. These properties implicate IL-11 as an additional multifunctional regulator in the hematopoietic microenvironment.