Dorsal-ventral limb patterning in vertebrates is thought to be controlled by the LIM-homeodomain protein Lmx1b which is expressed in a spatially and temporally restricted manner along the dorsal-ventral limb axis. Here we describe the phenotype resulting from targeted disruption of Lmx1b. Our results demonstrate that Lmx1b is essential for the specification of dorsal limb fates at both the zeugopodal and autopodal level with prominent phenotypes including an absence of nails and patellae. These features are similar to those present in a dominantly inherited human condition called nail patella syndrome (NPS), which also has renal involvement. Mouse Lmx1b maps to a region syntenic to that of the NPS gene, and kidneys of Lmx1b mutant mice exhibit pathological changes similar to that observed in NPS (refs 5,6). Our results demonstrate an essential function for Lmx1b in mouse limb and kidney development and suggest that NPS might result from mutations in the human LMX1B gene.

Abnormal regulation and expression of microRNAs (miRNAs) has been documented in various diseases including cancer. The miRNA let - 7 (MIRLET7) family controls developmental timing and differentiation. Let - 7 loss contributes to carcinogenesis via an increase in its target oncogenes and stemness factors. Let - 7 targets include genes regulating the cell cycle, cell signaling, and maintenance of differentiation. It is categorized as a tumor suppressor because it reduces cancer aggressiveness, chemoresistance, and radioresistance. However, in rare situations let - 7 acts as an oncogene, increasing cancer migration, invasion, chemoresistance, and expression of genes associated with progression and metastasis. Here, we review let - 7 function as tumor suppressor and oncogene, considering let - 7 as a potential diagnostic and prognostic marker, and a therapeutic target for cancer treatment. We explain the complex regulation and function of different let - 7 family members, pointing to abnormal processes involved in carcinogenesis. Let - 7 is a promising option to complement conventional cancer therapy, but requires a tumor specific delivery method to avoid toxicity. While let - 7 therapy is not yet established, we make the case that assessing its tumor presence is crucial when choosing therapy. Clinical data demonstrate that let - 7 can be used as a biomarker for rational precision medicine decisions, resulting in improved patient survival.

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

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Lmx1b, a member of the LIM homeodomain protein family, is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail-patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal limb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression. In this study, in situ hybridizations of Lmx1b on murine limb sections reveal strong expression in dorsal mesenchymal tissues (precursors of muscle, tendons, joints and patella) and, interestingly, also in anterior structures of the limb, explaining the anterior to posterior gradient of joint and nail dysplasia observed in NPS patients. Transfection studies showed that both the LIM domain-interacting protein, LDB1, and the helix-loop-helix protein, E47/shPan1, can regulate LMX1B action. While co--transfections of E47/shPan1 with LMX1B result in a synergistic effect on reporter activity, LDB1 down-regulated LMX1B-mediated transactivation irrespective of E47/shPan1. Mutant LMX1B proteins containing human mutations affecting each of the helices or the N-terminal arm of the homeodomain abolished transactivation, while LIM B and truncation mutations retained residual activity. These mutations fail to act in a dominant-negative manner on wild-type LMX1B in mixing studies, thereby supporting haploinsufficiency as the mechanism underlying NPS pathogenesis.

Localized breast cancer can be treated with lumpectomy and postoperative radiation therapy, also called breast conservation therapy, with an efficacy equivalent to that of mastectomy. Reports evaluating the effects of radiotherapy suggested that breast conservation therapy had "acceptable" cosmetic outcomes; thus, posttreatment evaluation for aesthetic impact has not been instituted as a standard of care. More recent reports have suggested that the effect of breast conservation therapy on aesthetic outcome is not minimal and that patients may benefit from reconstructive consultation. The purpose of this study was to measure objectively the aesthetic change in women who undergo breast conservation therapy and whether the extent of change is significant enough (objectively and subjectively) to warrant plastic surgery consultation. The authors evaluated 21 patients who had undergone breast conservation therapy. Eleven non-breast cancer patients seeking plastic surgery consultation were used as controls. Standardized five-view photographs (frontal, left and right lateral, and left and right lateral oblique views) were obtained. Patient photograph sets were compiled and evaluated independently by eight reviewers (four surgeons, two nurses, and two medical students). Reviewers evaluated the photographs using the breast asymmetry score (score range, 0 to 9) assessing breast size, ptosis, nipple-areola position, shape, scar appearance, contour deformity, and skin changes. The authors considered 2 SD above the control mean as significant. Breast conservation therapy patients also completed a 15-item questionnaire targeting objective and subjective data about treatment-related breast change. Breast conservation therapy patients had an average treatment-related asymmetry score of 1.93, with 35 percent demonstrating significant change as compared with controls. Although most patients (86 percent) were satisfied with the cancer treatment outcome, all patients noted asymmetry. The authors' data indicate that breast conservation therapy can cause significant asymmetry; thus, an option for plastic surgery consultation as part of the treatment protocol is warranted.

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