A practical guide to data management and sharing for biomedical laboratory researchers.

Effective data management and sharing have become increasingly crucial in biomedical research; however, many laboratory researchers lack the necessary tools and knowledge to address this challenge. This article provides an introductory guide into research data management (RDM), and the importance of FAIR (Findable, Accessible, Interoperable, and Reusable) data-sharing principles for laboratory researchers produced by practicing scientists. We explore the advantages of implementing organized data management strategies and introduce key concepts such as data standards, data documentation, and the distinction between machine and human-readable data formats. Furthermore, we offer practical guidance for creating a data management plan and establishing efficient data workflows within the laboratory setting, suitable for labs of all sizes. This includes an examination of requirements analysis, the development of a data dictionary for routine data elements, the implementation of unique subject identifiers, and the formulation of standard operating procedures (SOPs) for seamless data flow. To aid researchers in implementing these practices, we present a simple organizational system as an illustrative example, which can be tailored to suit individual needs and research requirements. By presenting a user-friendly approach, this guide serves as an introduction to the field of RDM and offers practical tips to help researchers effortlessly meet the common data management and sharing mandates rapidly becoming prevalent in biomedical research.

Identification of the gene causing mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive, neurodegenerative, lysosomal storage disorder characterized by psychomotor retardation and ophthalmological abnormalities including corneal opacities, retinal degeneration and strabismus. Most patients reach a maximal developmental level of 12?15 months. The disease was classified as a mucolipidosis following observations by electron microscopy indicating the lysosomal storage of lipids together with water-soluble, granulated substances. Over 80% of the MLIV patients diagnosed are Ashkenazi Jews, including severely affected and mildly affected patients. The gene causing MLIV was previously mapped to human chromosome 19p13.2-13.3 in a region of approximately 1 cM (ref. 7). Haplotype analysis in the MLIV gene region of over 70 MLIV Ashkenazi chromosomes indicated the existence of two founder chromosomes among 95% of the Ashkenazi MLIV families: a major haplotype in 72% and a minor haplotype in 23% of the MLIV chromosomes (ref. 7, and G.B., unpublished data). The remaining 5% are distinct haplotypes found only in single patients. The basic metabolic defect causing the lysosomal storage in MLIV has not yet been identified. Thus, positional cloning was an alternative to identify the MLIV gene. We report here the identification of a new gene in this human chromosomal region in which MLIV-specific mutations were identified.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Molecular basis of variant pseudo-hurler polydystrophy (mucolipidosis IIIC)

Mucolipidosis IIIC, or variant pseudo-Hurler polydystrophy, is an autosomal recessive disease of lysosomal hydrolase trafficking. Unlike the related diseases, mucolipidosis II and IIIA, the enzyme affected in mucolipidosis IIIC (N-Acetylglucosamine-1-phosphotransferase [GlcNAc-phosphotransferase]) retains full transferase activity on synthetic substrates but lacks activity on lysosomal hydrolases. Bovine GlcNAc-phosphotransferase has recently been isolated as a multisubunit enzyme with the subunit structure alpha(2)beta(2)gamma(2). We cloned the cDNA for the human gamma-subunit and localized its gene to chromosome 16p. We also showed, in a large multiplex Druze family that exhibits this disorder, that MLIIIC also maps to this chromosomal region. Sequence analysis of the gamma-subunit cDNA in patients from 3 families identified a frameshift mutation, in codon 167 of the gamma subunit, that segregated with the disease, indicating MLIIIC results from mutations in the phosphotransferase gamma-subunit gene. This is to our knowledge the first description of the molecular basis for a human mucolipidosis and suggests that the gamma subunit functions in lysosomal hydrolase recognition.

Mucolipidosis type IV: novel MCOLN1 mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish population.

The gene MCOLN1 is mutated in Mucolipidosis type IV (MLIV), a neurodegenerative, recessive, lysosomal storage disorder. The disease is found in relatively high frequency among Ashkenazi Jews due to two founder mutations that comprise 95% of the MLIV alleles in this population [Bargal et al., 2000]. In this report we complete the mutation analysis of Jewish and non-Jewish MLIV patients whose DNA were available to us. Four novel mutations were identified in the MCOLN1 gene of severely affected patients: two missense, T232P and F465L; a nonsense, R322X; and an 11-bp insertion in exon 12. The nonsense mutation (R322X) was identified in two unrelated patients with different haplotypes in the MCOLN1 chromosomal region, indicating a mutation hotspot in this CpG site. An in-frame deletion (F408del) was identified in a patient with unusual mild psychomotor retardation. The frequency of MLIV in the general Jewish Ashkenazi population was estimated in a sample of 2,000 anonymous, unrelated individuals assayed for the two founder mutations. This analysis indicated a heterozygotes frequency of about 1/100. A preferred nucleotide numbering system for MCOLN1 mutations is presented and the issue of a screening program for the detection of high-risk families in the Jewish Ashkenazi population is discussed.

Protooncogene expression in normal and psoriatic skin.

The expression of the c-myc, c-fos, c-jun, c-erbB, and c-Ha-ras protooncogenes was compared by Northern blot analysis of total RNA extracted from keratome biopsies of normal skin and psoriatic plaques. Isolation of intact RNA from frozen tissue required careful attention to technique during the early stages of extraction. Densitometric analysis revealed 1.5- to 2.5-fold elevations of c-myc transcript levels in lesional psoriatic relative to normal epidermis. Similar increases in cyclophilin and lipocortin II transcripts were also observed and may reflect characteristic differences in RNA preparations from normal and psoriatic epidermis. C-myc, c-jun, c-erbB, c-fos, and c-Ha-ras transcript levels were not significantly increased in lesional psoriatic epidermis when protooncogene mRNA levels were normalized to those of the cyclophilin or lipocortin genes. In contrast, transforming growth factor-alpha (TGF-alpha) transcripts were significantly increased (10- to 20-fold) with or without prior normalization. C-myc, c-fos, and c-jun transcripts were significantly induced over in vivo levels 2-4 h after organ culture of normal or psoriatic keratome biopsies, demonstrating that these genes can be highly expressed in the context of tissue injury. Our results suggest that overexpression of these protooncogenes per se is not central to the pathogenesis of psoriatic epidermal hyperplasia.

Heparin-binding epidermal-growth-factor-like growth factor activation of keratinocyte ErbB receptors Mediates epidermal hyperplasia, a prominent side-effect of retinoid therapy.

Sun-protected human skin was maintained in organ culture and treated with all-trans retinoic acid in the presence or absence of reversible or irreversible pharmacologic antagonists of c-erbB receptor tyrosine kinase activity. In the absence of these inhibitors, all-trans retinoic acid induced epidermal hyperplasia comparable to that induced in intact skin by all-trans retinol or all-trans retinoic acid itself. There was a strong correlation between inhibition of epidermal hyperplasia in organ culture and inhibition of epidermal-growth-factor-dependent keratinocyte growth in monolayer culture. In additional studies it was shown that all-trans retinoic acid could overcome the known inhibitory effects of calcium on expression of HB-EGF-like growth factor mRNA in organ-cultured skin. Further, it was shown that an antibody to HB-EGF-like growth factor inhibited retinoid-stimulated epidermal hyperplasia in organ culture and reduced proliferation in cultured keratinocytes. In contrast, the c-erbB receptor tyrosine kinase antagonists and the neutralizing HB-EGF-like growth factor antibody were ineffective in inhibiting all-trans-retinoic-acid-dependent survival and proliferation of human dermal fibroblasts. Taken together, these data indicate (i) that retinoid-induced epidermal hyperplasia in human skin proceeds through c-erbB, and (ii) that HB-EGF-like growth factor is one of the c-erbB ligands mediating this effect.

Mucolipidosis type IV: the origin of the disease in the Ashkenazi Jewish population.

Mucolipidosis type IV (MLIV) is a neurodegenerative lysosomal storage disease in which most of the patients diagnosed hitherto are Ashkenazi Jews. The basic metabolic defect causing this disease is still unknown and the relevant gene has not yet been mapped or cloned. Seventeen Israel Ashkenazi families with MLIV patients had been interviewed to study their family origin. Although the families immigrated to Israel from various European countries they all could trace their roots three to four generations back to northern Poland or the immediate neighbouring country, Lithuania. Furthermore, there are only one or two ultraorthodox families among the 70-80 Ashkenazi families with MLIV patients worldwide, a marked under-representation of this group which constitutes at least 10% of the Ashkenazi population. This data indicate that MLIV mutation occurred only around the 18th and 19th centuries, after the major expansion of this population, in a founder in this defined European region belonging to a more modern, secular family.

Adult-onset Niemann-Pick type C disease. Clinical, biochemical, and genetic study.

BACKGROUND: Niemann-Pick type C disease is an autosomal recessive neurometabolic disorder of unknown origin mapped to chromosome 18q11-12 in most of the studied families. In contrast to the sphingomyelin lipidoses, in Niemann-Pick type C disease, fibroblasts are impaired in intracellular homeostatic responses to exogenous low-density lipoprotein (LDL) cholesterol. Biochemical heterogeneity of the disorder in relation to abnormal LDL processing is associated with various clinical presentations, but adult-onset Niemann-Pick type C disease is rare and has not been comprehensively characterized. OBJECTIVE: To describe clinical, biochemical, and genetic features of adult-onset Niemann-Pick type C disease in 3 siblings. DESIGN AND SETTING: Case series in a tertiary care center. PATIENTS: The 3 siblings manifested a variable combination of vertical supranuclear ophthalmoplegia, ataxia, and splenomegaly. Brain magnetic resonance imaging showed cerebellar atrophy; brainstem auditory evoked responses were unobtainable, and bone marrow examination disclosed typical foam cells. The patients were 20, 26, and 28 years old and belonged to a sibship of 13 born of consanguineous healthy parents. METHODS: Esterification of exogenous LDL cholesterol in cultured skin fibroblasts and filipin staining for free intracellular cholesterol. Polymerase chain reaction-based DNA linkage study using AC microsatellite markers D18S40, D18S44, D18S480, and D18S66. RESULTS: Fibroblasts of the 3 patients showed a 23% to 58% block in the induced cholesterol esterification after 4 1/2 hours and a mild to moderate accumulation of free cholesterol. DNA study demonstrated linkage to the major 18q11-12 Niemann-Pick type C locus and identified unaffected carriers. CONCLUSIONS: These results confirm the diagnosis of the least biochemically affected Niemann-Pick type C phenotype in this family with adult-onset disease and support a correlation between the mild laboratory and clinical findings in this age group.

A cDNA from the ovarian cancer critical region of deletion on chromosome 17p13.3.

Chromosome 17p13.3 is frequently deleted in human ovarian carcinoma, and the 15 kb critical region of deletion may contain a tumor suppressor gene. A 2.3 kb cDNA has been identified which spans 17 kb of genomic DNA, including 8.1 kb within the critical region, and thus is a candidate tumor suppressor gene. This highly conserved gene has significant sequence similarity to a yeast gene of unknown function and to one of the yeast enzymes in the diphthamide synthetic pathway, DPH2, that has a role in global protein synthesis regulation. This gene, named DPH2L (diphthamide biosynthesis protein 2-like), is expressed in multiple tissues and stages of development.

Human squamous carcinoma cell invasion in organ-cultured skin.

In a previous study we showed that normal human epidermal keratinocytes were capable of invading the dermis of organ cultured skin when the tissue was treated with an exogenous source of epithelial growth factors (Fligiel and Varani (1993) Invasion Metastasis, 13, 225-233). Here we examined human squamous carcinoma cells from three different tumors for ability to invade the dermis in the same model. Invasion occurred in 40-80% of the tissues, depending on the tumor line, and was observed with equal frequency under both growth factor-free conditions and in the presence of exogenous growth factors. These data demonstrate that malignant human epithelial cells have the capacity to invade the dermis of organ-cultured skin. Unlike normal keratinocytes, there appears to be no exogenous growth factor requirement for invasion by the malignant cells.

Regulation of mammary differentiation by the extracellular matrix.

In multicellular organisms cell growth and differentiation are influenced by soluble factors, cell-cell interactions and cell-extracellular matrix interactions. We have used the rat mammary gland as a model system to study the role of extracellular matrix components in the regulation of milk protein gene expression. Since mammary epithelial cells differentiate on a basement membrane in vivo, we investigated the effects of basement membrane components on the expression of the milk protein genes, alpha-casein, alpha-lactalbumin, and transferrin. We have demonstrated that a basement membrane gel, as well as its major basement membrane component, laminin, induced alpha-casein and alpha-lactalbumin expression as much as 160-fold compared to tissue culture plastic. We demonstrate that laminin affects mRNA stability as well as having an effect on protein stability and secretion. Laminin interacts with mammary epithelial cells via an 68 kD cell surface receptor which is capable of interacting with the cellular cytoskeleton. In order to provide evidence that laminin affects on mammary differentiation are mediated through this receptor via the cytoskeleton, we examined the effects of cytoskeletal disrupting agents on milk protein gene expression. We demonstrate that cytochalasin D or colchicine selectively block laminin-mediated milk protein gene expression by affecting mRNA stability. Based on these experiments, we propose a model in which laminin affects mammary gene expression through interaction with cell surface receptors which interact with the cytoskeleton resulting in stabilization of mRNAs for milk protein genes.

Screening for carriers of Tay-Sachs disease in the ultraorthodox Ashkenazi Jewish community in Israel.

A screening program for the detection of Tay-Sachs disease (TSD) carriers in the ultra Orthodox community of Ashkenazi Jews has operated in Israel since 1986. The purpose of this program is the prevention of marriages of 2 heterozygotes. The screened individuals are mostly couples in the engagement process or students in religious high schools. Two mandatory requirements guide this program. First, anonymity of the tested individuals who are identified only by code numbers; second completion of the test results of couples in the engagement process within a few days. The screening program is performed by the determination of hexosaminidase A (Hex A) activity in serum which is repeated in serum and leukocyte extracts in couples where both partners were found in the heterozygote range in the initial tests. The minimal carrier frequency was estimated to be 1:26 or higher, which is higher then in the general Jewish Ashkenazi population. This higher carrier frequency apparently stems from the fact that most members of this community originate from central Europe where the TSD carrier frequency was previously reported to be the highest in the Ashkenazi population. Since the beginning of the screening program no TSD child has been born to newlywed couples of this community in Israel.

Gaucher disease type I and pregnancy.

We surveyed 47 pregnancies of 17 women affected with Gaucher disease (GD) type I. In two women affected with the severe form of GD type I, no change was observed in the course of the disease during pregnancy. In one patient with the moderate form of the disease there was an exacerbation of the disease during and after pregnancy, and thereafter two subsequent pregnancies of this woman ended by early spontaneous abortion. Four women were diagnosed during their pregnancy or soon after delivery suggesting in these women an exacerbation related to pregnancy. In the other ten women there was no change in the course of the disease. In general, the pregnancies of women affected with GD were normal; however, six women needed blood transfusion during pregnancy or at delivery. From these data it is suggested that there is some risk to pregnant women affected with GD type I, and accordingly, appropriate follow-up should be planned at the beginning of pregnancy in these patients.

Krabbe disease: increased incidence in a highly inbred community.

Krabbe disease (globoid cell leukodystrophy) was found with very high incidence (6/1,000 live births) in a large Druze kindred in Israel. The clinical data on 12 of the affected children demonstrated clinical variability even though these children are homozygous for the same mutation by descent from a common ancestor.

Nortriptyline hydrochloride in urology.

Forty female patients suffering for long periods from frequency, urgency, and dysuria without any definite organic cause received nortriptyline chloride for three weeks. In 75 percent there was either considerable improvement or total disappearance of urinary complaints. At the same time the in vitro effects of the drug were tested using the isometric muscle contraction technique. Nortriptyline was found to have anticholinergic properties. The importance of this effect in the clinical study and the possible mode of action of the drug are discussed.

Troglitazone improves psoriasis and normalizes models of proliferative skin disease: ligands for peroxisome proliferator-activated receptor-gamma inhibit keratinocyte proliferation.

BACKGROUND: Psoriasis is often treated with agents that activate nuclear hormone receptors for glucocorticoids, retinoids, and vitamin D. The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a related nuclear hormone receptor that can be activated by its ligands, including the thiazolidinediones. OBJECTIVE: To assess whether treatment with troglitazone, a currently available thiazolidinedione used to treat diabetes mellitus, has an effect on psoriasis in normoglycemic patients and whether ligands for PPARgamma have an effect on models of psoriasis. DESIGN: Open-label administration of troglitazone in patients with psoriasis and evaluation of drug actions in cellular, organ, and transplant models of psoriasis. SETTING: University and community hospital outpatient departments and university laboratories. PATIENTS: Patients with chronic, stable plaque psoriasis and control subjects. Five patients with psoriasis received troglitazone (none withdrew); 10 different untreated patients and 10 controls provided tissue samples. INTERVENTIONS: Oral troglitazone therapy at various dosages in patients with psoriasis; also, use of troglitazone, ciglitazone, and 15-deoxy-delta-12,14-prostaglandinJ2 in psoriasis models. MAIN OUTCOME MEASURES: Investigator-determined clinical results in patients and cell counts and histological evidence in models. RESULTS: All patients' psoriasis improved substantially during troglitazone therapy. Peroxisome proliferator-activated receptor-gamma was expressed in human keratinocytes; ligands for PPARgamma inhibited the proliferation of normal and psoriatic human keratinocytes in culture. Troglitazone treatment normalized the histological features of psoriatic skin in organ culture and reduced the epidermal hyperplasia of psoriasis in the severe combined immunodeficient mouse and human skin transplant model of psoriasis (P<.05 compared with untreated controls). CONCLUSIONS: Peroxisome proliferator-activated receptor-gamma might be a useful intracellular target for the treatment of psoriasis; further study is needed to assess the clinical value of ligands for PPARgamma, including troglitazone.

Internalization of exogenous gangliosides in cultured skin fibroblasts for the diagnosis of mucolipidosis IV.

The internalization of exogenous mixed brain gangliosides in ML IV cultured skin fibroblasts indicated an impairment of ganglioside catabolism in these cells. Incubation of ML IV, normal and various other lysosomal storage disorders cell lines for five days with exogenous tritium labelled GM3, GD1a or GT1 gangliosides allowed accurate quantitation of the retained gangliosides. This in vitro approach provides a reliable method for the diagnosis of ML IV.

Biochemical investigations of cultured amniotic fluid cells in mucolipidosis type IV.

Biochemical abnormalities similar to those observed in cultured fibroblasts of patients with mucolipidosis type IV were demonstrated in cultured amniotic fluid cells of two fetuses affected with mucolipidosis IV. Increased gangliosides and acid mucopolysaccharides were observed in the affected cultures when compared to two normal controls. Both GM3 (monosialo) and GD3 (disialo) gangliosides accumulated in the affected cells: the latter showing a three-fold and the former a two-fold increase over controls. The major mucopolysaccharide components were dermatan sulfate and heparan sulfate, both increased approximately four-fold. A partial, but significant deficiency of soluble ganglioside sialidase was observed in the two affected cultures, while this activity was normal in a culture of a non-affected fetus of the same mother in a third pregnancy. Non-soluble membrane-bound and neuraminlactose sialidase was not affected.

Prenatal diagnosis of Krabbe disease using a fluorescent derivative of galactosylceramide.

A fluorescent substrate 12-(N-methyl-N(7-nitro-2-oxa-1,3-diazol-4-yl) aminododecanoyl sphingosyl beta-D-galactoside ('NBD galactocerebroside') was synthesized and used for the detection of galactocerebrosidase activity. The enzyme determinations using this substrate were found to be extremely sensitive yielding unambiguous results. This substrate was used for the prenatal diagnosis of a fetus affected with Krabbe disease; the diagnosis was later confirmed in the aborted fetus.