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Background and objectives Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. Design, setting, participants, & measurements Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to Results In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1, PLCE1, NPHS2, and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. Conclusions Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome.

No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets., Nephrotic syndrome is the second most common chronic kidney disease but there are no targeted treatment strategies available. Here the authors identify mutations of six genes codifying for proteins involved in cytoskeleton remodelling and modulation of small GTPases in 17 families with nephrotic syndrome.

Background and Purpose— Childhood arterial ischemic stroke is frequently associated with an intracranial arteriopathy that often progresses in the first 3 to 6 months post stroke. We hypothesized that children with enhancing arteriopathies on vessel wall imaging (VWI) would have a higher risk of arteriopathy progression than those without enhancement. Methods— Our institutional radiographic database was searched for cases of childhood stroke with VWI. Inclusion criteria consisted of age ranging from 1 month through 20 years, diagnosis of arterial ischemic stroke, available VWI, and follow-up magnetic resonance angiogram. Imaging was reviewed to systematically describe VWI findings, categorize arteriopathies, steroid therapy, and identify progressive arteriopathies using CACADE definitions. Results— Sixteen cases of childhood stroke at Children’s Hospital Colorado between January 1, 2010 and July 1, 2016 were reviewed. Strong vessel wall enhancement at presentation was associated with progressive arteriopathy in 83% of cases (10/12), when compared with 0% (0/4) without strong enhancement ( P =0.008). Conclusions— Our case series demonstrates the potential benefit of VWI in children with stroke because it may identify patients who will have progressive arterial disease.

Galloway-Mowat syndrome (GAMOS) is a severe autosomal-recessive disease characterized by the combination of early-onset steroid-resistant nephrotic syndrome (SRNS) and microcephaly with brain anomalies. To date, mutations of WDR73 are the only known monogenic cause of GAMOS and in most affected individuals the molecular diagnosis remains elusive. We here identify recessive mutations of OSGEP, TP53RK, TPRKB, or LAGE3, encoding the 4 subunits of the KEOPS complex in 33 individuals of 30 families with GAMOS. CRISPR/Cas9 knockout in zebrafish and mice recapitulates the human phenotype of microcephaly and results in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibits cell proliferation, which human mutations fail to rescue, and knockdown of either gene activates DNA damage response signaling and induces apoptosis. OSGEP and TP53RK molecularly interact and co-localize with the actin-regulating ARP2/3 complex. Furthermore, knockdown of OSGEP and TP53RK induces defects of the actin cytoskeleton and reduces migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identify 4 novel monogenic causes of GAMOS, describe the first link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.

Transabdominal ultrasound is the imaging modality of choice in the evaluation of girls with pathologic ovarian conditions such as ovarian masses, ovarian torsion or hemorrhagic ovarian cysts. The shape of the ovaries and their structure, size and blood flow can be determined by ultrasound. Before puberty, the ovaries have a volume of about 1 ml and contain small, non-stimulated follicles (I.J. Griffin et al. Acta paediatrica 1995; 84: 536–543). The presence of unilateral ovarian enlargement in a prepubertal girl may be a sign of torsion or of an ovarian mass and usually warrants further radiological evaluation or laparoscopic exploration (M.F. Brown et al. J Pediatr Surg 1993; 28: 930–933). The aim of this case report is to describe the value of abdominal ultrasound in unilateral ovarian stimulation in a prepubertal female patient with perforated appendicitis.

Nucleoporins are essential components of the nuclear pore complex (NPC). Only a few diseases have been attributed to NPC dysfunction. Steroid-resistant nephrotic syndrome (SRNS), a frequent cause of chronic kidney disease, is caused by dysfunction of glomerular podocytes. Here we identify in eight families with SRNS mutations in NUP93, its interaction partner NUP205 or XPO5 (encoding exportin 5) as hitherto unrecognized monogenic causes of SRNS. NUP93 mutations caused disrupted NPC assembly. NUP93 knockdown reduced the presence of NUP205 in the NPC, and, reciprocally, a NUP205 alteration abrogated NUP93 interaction. We demonstrate that NUP93 and exportin 5 interact with the signaling protein SMAD4 and that NUP93 mutations abrogated interaction with SMAD4. Notably, NUP93 mutations interfered with BMP7-induced SMAD transcriptional reporter activity. We hereby demonstrate that mutations of NUP genes cause a distinct renal disease and identify aberrant SMAD signaling as a new disease mechanism of SRNS, opening a potential new avenue for treatment.

In a prospective study between July 1999 and September 2000, stool specimens of children below the age of 16 years with ( n = 187) and without ( n = 137) diarrhea were tested for the presence of enterovirulent bacteria by standard culture methods and by PCR. Targets for the PCR were the plasmid pCVD432 for enteroaggregative Escherichia coli (EAEC), the verotoxin 1 and verotoxin 2 genes for enterohemorrhagic E. coli , ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., genes coding for heat-stable and heat-labile toxins for enterotoxigenic E. coli (ETEC), and the eaeA gene for enteropathogenic E. coli. The following bacteria could be associated with diarrhea: Salmonella enterica ( P = 0.001), Campylobacter spp. ( P = 0.036), ETEC ( P = 0.012), and EAEC ( P = 0.006). The detection of EAEC, ETEC, and S. enterica was strongly associated with a history of recent travel outside of Switzerland. EAEC isolates were found in the specimens of 19 (10.2%) of 187 children with diarrhea and in those of 3 (2.2%) of 137 children without diarrhea ( P = 0.006) and were the most frequently detected bacteria associated with diarrhea. Among the children below the age of 5 years, the specimens of 18 (11.9%) of 151 with diarrhea were positive for EAEC, while this agent was found in the specimens of 2 (2.2%) of 91 controls ( P = 0.007). Enteropathogenic E. coli isolates were found in the specimens of 30 (16.4%) of the patients and in those of 15 (10.9%) of the controls, with similar frequencies in all age groups ( P > 0.05). We conclude that EAEC bacteria are involved in a significant proportion of diarrhea cases among children. Children younger than 5 years of age are more often affected by EAEC than older children.

Issued US patents are utilised to describe the history of tissue engineering by implantation of dissociated cells in polymeric matrices. The history begins with patents issuing on applications filed in the late 1970s by Drs Yannas and Bell, who later founded Organogenesis. Next, the pivotal role of the technology developed by Drs Joseph Vacanti and Robert Langer is discussed, then the stromal cell matrices of Dr Gail Naughton, who founded Advance Tissue Sciences. The formation of businesses and product development is briefly correlated with the patents. Additional developments as described in the patents relate to methods for making polymeric matrices, methods for modifying polymers to interact with and modify the cellular interactions with the matrices, including cell attachment, differentiation and proliferation, and more recent advances specific to developments with stem cells and genetically engineered cells.

Nephrotic syndrome (NS), the association of gross proteinuria, hypoalbuminaemia, edema, and hyperlipidemia, can be clinically divided into steroid-sensitive (SSNS) and steroid-resistant (SRNS) forms. SRNS regularly progresses to end-stage renal failure. By homozygosity mapping and whole exome sequencing, we here identify recessive mutations in Crumbs homolog 2 (CRB2) in four different families affected by SRNS. Previously, we established a requirement for zebrafish crb2b, a conserved regulator of epithelial polarity, in podocyte morphogenesis. By characterization of a loss-of-function mutation in zebrafish crb2b, we now show that zebrafish crb2b is required for podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking. Furthermore, by complementation experiments in zebrafish, we demonstrate that CRB2 mutations result in loss of function and therefore constitute causative mutations leading to NS in humans. These results implicate defects in podocyte apico-basal polarity in the pathogenesis of NS.

In this secondary analysis, 890 patients 40 years of age and older were assessed within 24 hours of transfer from intensive care to medical/surgical units. Nursing diagnoses documented in the hospital record were compared to supporting clinical evidence obtained from subjects, the hospital record, and interviews with subjects and nurse caregivers. The congruence, based on the kappa statistic, between the nursing diagnosis and clinical evidence ranged from 0.1% for altered thought processes to 10% for impaired gas exchange, impaired physical mobility, and potential for injury, while no diagnoses were made for self-care deficits or sensory alterations, visual, when clinical evidence for them was available.

Background: Atypical haemolytic uremic syndrome (aHUS) is often associated with a high risk of disease recurrence and subsequent graft loss after isolated renal transplantation. Evidence-based recommendations for a mutation-based management after renal transplantation in aHUS caused by a combined mutation with complement factor I (CFI) and membrane cofactor protein CD46 (MCP) are limited. Case-diagnosis/Treatment: We describe a 9-year-old boy with a first manifestation of aHUS at the age of 9months carrying combined heterozygous mutations in the CFI and MCP genes. At the age of 5years, he underwent isolated cadaveric renal transplantation. Fresh frozen plasma was administered during and after transplantation, tapered and finally stopped after 3years. Conclusions: During the 5-year follow-up after transplantation there have been no signs of aHUS recurrence and graft function has remained good. The combination of heterozygous MCP and CFI mutations with aHUS might have a positive impact on the post-transplant course, possibly predicting a lower risk of aHUS recurrence after an isolated cadaveric renal transplantation

FOR THE BIO-ASSAY of adrenal cortical hormone preparations the survival-growth test (Cartland and Kuizenga, 1936) is of value, since it is responsive to the composite activity of adrenal cortex extracts with respect to both the known compounds and amorphous fractions (Kuizenga, Nelson and Ingle, 1943; and Kuizenga, Nelson, Lyster and Ingle, 1945). Factors influencing electrolyte and carbohydrate metabolism cannot, however, be differentiated by this test. Responses obtained with the pure C-11-oxygenated steroids in the muscle work test, compared with the relative inactivity of desoxycorticosterone and the amorphous fractions by this test, led us to the conclusion that it could be used for the quantitative determination of the specific factors effective in carbohydrate metabolism (Ingle, 1944; and Ingle and Kuizenga, 1945). For clinical use, a knowledge of the content of these factors in adrenal cortical extracts is of importance (MacBryde and De La Baize, 1944; and Forsham, Thorn and Bergner, 1946). We hav...

EFFECTIVE NOVEMBER 29, 2000, the U.S. Patent and Trademark Office (PTO) changed its policy of preventing the public from obtaining information about patent applications. Patent applications that claim priority to applications filed after November 29, 2000 will now be published 18 months after their earliest filing date for which they claim priority. This change in U.S. patent law helps the United States conform with trade agreements it made in the 1990s (i.e., the General Agreement on Tariffs and Trade and TradeRelated Aspects of Intellectual Property Agreement [GATT/TRIPs]). There are exceptions to the new rule regarding publication. The 18-month publication rule does not apply to (1) design applications, (2) provisional applications, (3) express requests by an applicant to not publish the application along with a certification that the application is not being filed in another country which requires 18-month publication, and (4) applications that are subject to a secrecy order. Applicants who are only filing in the United States or are filing in another country which does not abide by the 18-month publication rule may prevent publication by promptly filing a request that the application not be published. However, if the application is later filed in Europe or some other country that requires publication after 18 months, the applicants must inform the PTO of this change within 45 days of filing in the other country, or risk losing their U.S. application. Although the rule provides that provisional applications are not published, if an utility application claims priority to a provisional application, and the application issues as a U.S. Patent, then the provisional application will become available as part of the file history of the patent. This change in U.S. patent law has a number of implications. First, it potentially increases one’s awareness of what one’s colleagues and competitors are developing by publishing patent applications and allowing one to request copies of the file histories for published applications from the PTO before a patent issues. This represents a drastic change in U.S. patent law. Second, it provides applicants with a right to obtain a reasonable royalty during the period beginning on the date of publication and ending on the date the patent actually issues (see 35 U.S.C. § 154 (d)). Third, it may expedite the patent prosecution process by forcing applicants to comply with formality requirements for patent applications at the beginning of prosecution. However, on a practical basis, where most significant applications are being foreign filed, typically through the PCT, and published at 18 months, this change makes little difference with respect to availability of the information. The significant change is with regard to the potential right to obtain royalties for the period between publication and issuance. A key factor in obtaining royalties will be whether or not the product which is sold (and for which royalties are therefore due) is adequately described and claimed in the published application.

IN A CHANGE to conform U.S. patent practice to practice in Europe, Asia and most other countries, U.S. patent applications filed November, 29, 2000, and thereafter will be published 18 months after the effective filing date of the application (subject to applicants’ attestation upon filing that they have not and will not file an application for the same invention in a foreign country or under a multilateral international agreement, that requires publication of applications 18 months after filing). For those applications which are being foreign filed, this will not have any impact. However, in the technical fields where only U.S. rights are typically pursued, this could result in a significant change to the number of patent applications being filed. In another area of U.S. patent practice, investigators need to be aware that keeping records on computers, rather than in laboratory notebooks or as hard copy printouts, may result in loss of patent rights should there be a priority contest. Outside of the U.S., most countries and regional patent offices (such as the European Patent Office) award patent rights to the party who is first to file, regardless of when the technology was invented. In the U.S., however, rights go to the party who is first to invent, barring intervening prior art that discloses the invention more than one year prior to the second party to file. When two or more parties claim the same invention, the U.S. patent office will declare an “interference,” an inter pertes proceedings to determine who was first to invent, and therefore who is entitled to the patent. In the biotechnology field, applications may be filed within a very short time frame, even a matter of one to two weeks. The contesting parties are then required to prove who conceived and reduced to practice the invention first. Traditionally, this has been done by submitting copies of laboratory notebooks, reports, draft publications, grant applications, and copies of other data, which is then “corroborated” by the testimony of non-inventors. In view of the changes to record keeping by so many scientists (especially those in research institutions and companies without in house patent counsel), this may become more problematic. Many scientists now keep all of their data on computers in files which they regularly update. This creates two problems. The first is that there is no evidence of when data is entered into the file, so it becomes impossible to verify the date of invention. Second, because the dates on computers can be changed so easily, the Patent Office (and courts) will not accept computer printouts as evidence absent other corroborating evidence. Accordingly, for those working in highly competitive fields, we recommend printing out copies of data on a regular basis (once a month at most; more frequently during critical times), having the print outs signed and dated by the inventors and witnessed by at least one non-inventor, then keeping the hard copies in notebooks or files. Since interferences usually are declared long after the invention is made (not uncommonly four to eight years after the date the application for patent is first filed), obtaining corroborating evidence will be extremely difficult and rights may be awarded to the other party.

Tissue engineering involves a number of complex legal issues. Those that will be addressed in this chapter are limited to intellectual property issues and interactions with the Food and Drug Administration in the United States, with a brief discussion of corresponding intellectual property issues outside of the United States.

As we begin this new millennium, we are confronted with emerging technologies in the field of biomedical engineering that broaden the horizon of medical treatment by implantation. For example, research scientists are currently developing a neural arm prosthesis that will stimulate sensations corresponding to the amounts of pressure that a person would normally feel in their hand when doing various tasks.1 In another example, other inventors have developed a process by which a patient’s own cartilage can be used to create new tissue that can be surgically implanted to replace the damaged tissue.2 Each year millions of people depend on the availability of such life-saving or life-enhancing medical implantation devices. In turn, many of the designers, manufacturers, and sellers of these life-saving or life-enhancing medical implantation devices depend on a continued supply of raw materials and component parts for the development, improvement, and maintenance of these devices. What would happen if the supply of raw materials and component parts used in these medical implantation devices suddenly stopped? The medical device industry has been threatened with just such a possibility. The raw materials and components suppliers were being subjected to huge litigation expenses in defending a vast amount of, and often frivolous, lawsuits from plaintiffs who alleged that the suppliers’ materials harmed them in some way. Such allegations included the inadequate design and testing or inadequate warning of medical implantation devices manufactured with the materials or parts provided by the suppliers. Although it was very rare for a supplier to actually be held liable in these actions, the economic costs involved in defending such lawsuits created a lack of economic incentive for the suppliers to continue to sell their products to medical device companies for implantable purposes. As a result of these lawsuits, the medical industry was faced with the very real possibility that the production of these life-saving or lifeenhancing devices would have to cease. In an effort to address the concerns facing both the medical industry and the raw materials and component parts industry, President Clinton, on August 13, 1998, signed into law a bill entitled the “Biomaterials Access Assurance Act.”3 This law provides for expeditious procedures to dispose of unwarranted lawsuits against the raw materials and component parts suppliers in a manner as to minimize litigation costs. Examples of such expeditious procedures include allowing suppliers to move for motions to dismiss or motions for summary judgments. However, what if there is a situation where the raw materials and component parts supplier is truly at fault? Don’t fret, this law does not automatically dispose of all lawsuits brought by plaintiffs against the supplier, just those that are without merit. Those lawsuits with merit include actions for harm to a claimant caused by an implant if the supplier is also the manufacturer of the implant; actions for harm to a claimant caused by an implant if the supplier is also the seller of the implant; actions

IT HAS BEEN reported by Ingle (1940a, 1940b) that 11-desoxycorticosterone and its acetate were less active than the C-11-oxygenated adrenal cortical steroids in sustaining the ability of the adrenalectomized rat to work. According to VOgtli (1943a, 1943b) the apparent ineffectiveness of 11-desoxycorticosterone was due solely to its slowabsorption and animals treated for several days prior to the beginning of the work test performed as much work as did normal animals. Vogtli omitted consideration of the results of Ingle (1940b) who compared the work performance of adrenalectomized rats treated postoperatively for one week with 11-desoxycorticosterone and its acetate with the effects of 17-hydroxy-ll-dehydrocorticosterone and its acetateand found that under these conditions also the C-11-oxygenated compounds were muoh more potent than the 11-desoxy compounds. VIT HAS BEEN reported by Ingle (1940a, 1940b) that 11-desoxycorticosterone and its acetate were less active than the C-11-oxygenated adrenal cortical ...

Assay data on the U. S. P. XIV digitalis reference standard, obtained over a four- year period, have been examined statistically to show that a seasonal variation exists in the response of pigeons to the reference standard. Investigation was made of the effect of seasonal variation on relative potency by the comparison of data obtained at thirty-day and sixty-day intervals. It was found that the seasonal variation had little effect if the U. S. P. XIV assay completion limit of thirty days was observed. Greater variation occurred when the time interval was increased to sixty days.

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The stability of crude digitalis leaf was studied for two years, using the U. S. P. XIV pigeon method of assay. The data showed: (a) a loss of potency in the first twelve months, (b) no difference in stability between the ground or whole leaf forms, and (c) possibly greater stability when storage was in sealed rather than unsealed containers.