Reduced Abd-B Hox function during kidney development results in lineage infidelity.

Hox genes can function as key drivers of segment identity, with Hox mutations in Drosophila often resulting in dramatic homeotic transformations. In addition, however, they can serve other essential functions. In mammals, the study of Hox gene roles in development is complicated by the presence of four Hox clusters with a total of 39 genes showing extensive functional overlap. In this study, in order to better understand shared core Hox functions, we examined kidney development in mice with frameshift mutations of multiple Abd-B type Hox genes. The resulting phenotypes included dramatically reduced branching morphogenesis of the ureteric bud, premature depletion of nephron progenitors and abnormal development of the stromal compartment. Most unexpected, however, we also observed a cellular level lineage infidelity in nephron segments. Scattered cells within the proximal tubules, for example, expressed genes normally expressed only in collecting ducts. Multiple combinations of inappropriate nephron segment specific marker expression were found. In some cases, cells within a tubule showed incorrect identity, while in other cases cells showed ambiguous character, with simultaneous expression of genes associated with more than one nephron segment. These results give evidence that Hox genes have an overlapping core function at the cellular level in driving and/or maintaining correct differentiation decisions.

Coastal Tourism and Its Influence on Wastewater Nitrogen Loading: A Barrier Island Case Study.

Package treatment plants (PTPs) are facilities designed to treat onsite wastewater for small communities, commercial, and residential developments. PTPs are being utilized in a growing number of coastal communities. This study estimated the effects of coastal tourism on onsite wastewater nitrogen (N) inputs to a barrier island surficial aquifer (Bogue Banks, NC). The N-removal effectiveness was assessed for seven PTPs that treated wastewater from vacation properties using a range of technologies: extended aeration; sequencing batch reactor; and advanced media filtration. Influent and effluent wastewater samples were collected monthly from Feb. 2014 to Jan. 2015 and analyzed for particulate and dissolved N. Increased summer visitation associated with coastal tourism resulted in an increase in water use, wastewater inputs, and PTP N loading to the surficial aquifer. However, extended aeration systems did not have significantly elevated TN loads during the summer months because their treatment efficiency increased. N inputs associated with coastal tourism made up approximately 51% of the annual wastewater-related N load to the surficial aquifer. Onsite wastewater N-loading to the surficial aquifer (6.7 kg-N/ha/yr) appeared to be the dominant source of N loading on the island. Water quality data indicated that these N inputs have resulted in increased groundwater NO3 concentrations in the surficial aquifer. Overall, wastewater inputs added approximately 4.6 cm of groundwater recharge annually to the island. Coastal tourism can result in measurable increases in wastewater N loading, groundwater nitrogen concentrations, and groundwater recharge.

Towards more tolerable subcutaneous administration: Review of contributing factors for improving combination product design.

Subcutaneous (SC) injections can be associated with local pain and discomfort that is subjective and may affect treatment adherence and overall patient experience. With innovations increasingly focused on finding ways to deliver higher doses and volumes (≥2 mL), there is a need to better understand the multiple intertwined factors that influence pain upon SC injection. As a priority for the SC Drug Development & Delivery Consortium, this manuscript provides a comprehensive review of known attributes from published literature that contribute to pain/discomfort upon SC injection from three perspectives: (1) device and delivery factors that cause physical pain, (2) formulation factors that trigger pain responses, and (3) human factors impacting pain perception. Leveraging the Consortium's collective expertise, we provide an assessment of the comparative and interdependent factors likely to impact SC injection pain. In addition, we offer expert insights and future perspectives to fill identified gaps in knowledge to help advance the development of patient-centric and well tolerated high-dose/high-volume SC drug delivery solutions.

Preclinical Pharmacokinetic Study on Caffeine as an Excipient for Monoclonal Antibody Formulations.

Caffeine is a novel excipient that effectively reduces viscosity of high concentration mAb formulations intended for subcutaneous (SQ) delivery. Two preclinical studies were conducted in rats to evaluate pharmacokinetic (PK) parameters of caffeine as well as its effects on the PK profile of a model mAb, namely ipilimumab. Results show that SQ absorption and elimination of caffeine was rapid, with the average Tmax of 0.4 h and T1/2 of 1.6 h, administered with or without ipilimumab. Furthermore, caffeine did not affect ipilimumab SQ PK profiles. Independent of caffeine concentration, ipilimumab serum T1/2 was between 2 and 3 days, Tmax was between 3 and 4 days and SQ bioavailability was about 64%. In addition, SQ injection of caffeine at different dose levels showed no irritation at the injection site or adverse effects. Results from the current PK studies warrant further development of caffeine as a viscosity reducing excipient for mAb SQ formulations.

Caffeine as a Viscosity Reducer for Highly Concentrated Monoclonal Antibody Solutions.

Many monoclonal antibody (mAb) solutions exhibit high viscosity at elevated concentrations, which prevents manufacturing and injecting of concentrated mAb drug products at the small volumes needed for subcutaneous (SC) administration. Addition of excipients that interrupt intermolecular interactions is a common approach to reduce viscosity of high concentration mAb formulations. However, in some cases widely used excipients can fail to lower viscosity. Here, using infliximab and ipilimumab as model proteins, we show that caffeine effectively lowers the viscosity of both mAb formulations, whereas other common viscosity-reducing excipients, sodium chloride and arginine, do not. Furthermore, stability studies under accelerated conditions show that caffeine has no impact on stability of lyophilized infliximab or liquid ipilimumab formulations. In addition, presence of caffeine in the formulations does not affect in vitro bioactivities of infliximab or ipilimumab. Results from this study suggest that caffeine could be a useful viscosity reducing agent that complements other traditional excipients and provides viscosity reduction to a wider range of mAb drug products.

Single cell RNA-seq study of wild type and Hox9,10,11 mutant developing uterus.

The uterus is a remarkable organ that must guard against infections while maintaining the ability to support growth of a fetus without rejection. The Hoxa10 and Hoxa11 genes have previously been shown to play essential roles in uterus development and function. In this report we show that the Hoxa9,10,11, Hoxc9,10,11, Hoxd9,10,11 genes play a redundant role in the formation of uterine glands. In addition, we use single cell RNA-seq to create a high resolution gene expression atlas of the developing wild type mouse uterus. Cell types and subtypes are defined, for example dividing endothelial cells into arterial, venous, capillary, and lymphatic, while epithelial cells separate into luminal and glandular subtypes. Further, a surprising heterogeneity of stromal and myocyte cell types are identified. Transcription factor codes and ligand/receptor interactions are characterized. We also used single cell RNA-seq to globally define the altered gene expression patterns in all developing uterus cell types for two Hox mutants, with 8 or 9 mutant Hox genes. The mutants show a striking disruption of Wnt signaling as well as the Cxcl12/Cxcr4 ligand/receptor axis.

Disruption of Hox9,10,11 function results in cellular level lineage infidelity in the kidney.

Hox genes are important regulators of development. The 39 mammalian Hox genes have considerable functional overlap, greatly confounding their study. In this report, we generated mice with multiple combinations of paralogous and flanking Abd-B Hox gene mutations to investigate functional redundancies in kidney development. The resulting mice developed a number of kidney abnormalities, including hypoplasia, agenesis, and severe cysts, with distinct Hox functions observed in early metanephric kidney formation and nephron progenitor maintenance. Most surprising, however, was that extensive removal of Hox shared function in these kidneys resulted in cellular level lineage infidelity. Strikingly, mutant nephron tubules consisted of intermixed cells with proximal tubule, loop of Henle, and collecting duct identities, with some single cells expressing markers associated with more than one nephron segment. These results indicate that Hox genes are required for proper lineage selection/maintenance and full repression of genes involved in cell fate restriction in the developing kidney.

Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community.

Database URL: https://citrusgreening.org/.

Systemic amyloidosis associated with pleomorphic sarcoma of the spleen and remission of nephrotic syndrome after removal of the tumor.

We report a case of a 57-year-old woman who was diagnosed with a systemic AA amyloidosis associated with a pleomorphic sarcoma of the spleen. Although the association and causality between chronic inflammatory states and systemic AA amyloidosis have been well established, the evidence linking solid malignancies to reactive AA amyloidosis is scarce. Our patient had a significant systemic amyloid deposition including biopsy-proven renal and cardiac AA amyloidosis. Subsequent evaluation uncovered the presence of a large splenic mass, which was treated by splenectomy. Histologically the splenic tumor was classified as pleomorphic sarcoma. The removal of the tumor resulted in a marked decline in proteinuria, stable renal and cardiac functions, and symptomatic improvement at 1-year follow-up. Based on the noted improvements, we speculate that the pleomorphic sarcoma of the spleen caused secondary amyloidosis in our case. Stromal tumors, although rare, may be associated with and should be considered in the differential diagnosis of the cause of AA amyloidosis.

Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2.

The mammalian target of rapamycin (mTOR) is a major component of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway that is dysregulated in 50% of all human malignancies. Rapamycin and its analogues (rapalogs) partially inhibit mTOR through allosteric binding to mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report WYE-125132 (WYE-132), a highly potent, ATP-competitive, and specific mTOR kinase inhibitor (IC(50): 0.19 +/- 0.07 nmol/L; >5,000-fold selective versus PI3Ks). WYE-132 inhibited mTORC1 and mTORC2 in diverse cancer models in vitro and in vivo. Importantly, consistent with genetic ablation of mTORC2, WYE-132 targeted P-AKT(S473) and AKT function without significantly reducing the steady-state level of the PI3K/PDK1 activity biomarker P-AKT(T308), highlighting a prominent and direct regulation of AKT by mTORC2 in cancer cells. Compared with the rapalog temsirolimus/CCI-779, WYE-132 elicited a substantially stronger inhibition of cancer cell growth and survival, protein synthesis, cell size, bioenergetic metabolism, and adaptation to hypoxia. Oral administration of WYE-132 to tumor-bearing mice showed potent single-agent antitumor activity against MDA361 breast, U87MG glioma, A549 and H1975 lung, as well as A498 and 786-O renal tumors. An optimal dose of WYE-132 achieved a substantial regression of MDA361 and A549 large tumors and caused complete regression of A498 large tumors when coadministered with bevacizumab. Our results further validate mTOR as a critical driver for tumor growth, establish WYE-132 as a potent and profound anticancer agent, and provide a strong rationale for clinical development of specific mTOR kinase inhibitors as new cancer therapy.