Inhibiting centrosome clustering reduces cystogenesis and improves kidney function in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure, yet therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering," a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with 2 inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, decreased interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

Aberrant centrosome biogenesis disrupts nephron and collecting duct progenitor growth and fate resulting in fibrocystic kidney disease.

Mutations that disrupt centrosome biogenesis or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet how centrosome dysfunction results in the kidney disease phenotypes remains unknown. Here, we examined the consequences of conditional knockout of the ciliopathy gene Cep120, essential for centrosome duplication, in the nephron and collecting duct progenitor niches of the mouse embryonic kidney. Cep120 loss led to reduced abundance of both cap mesenchyme and ureteric bud populations, due to a combination of delayed mitosis, increased apoptosis and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis and decline in kidney function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in the pathways essential for development, fibrosis and cystogenesis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney morphogenesis and identifies new therapeutic targets for patients with renal centrosomopathies.

Evaluation of Candidate Theranostics for 227Th/89Zr Paired Radioimmunotherapy of Lymphoma.

227Th is a promising radioisotope for targeted α-particle therapy. It produces 5 α-particles through its decay, with the clinically approved 223Ra as its first daughter. There is an ample supply of 227Th, allowing for clinical use; however, the chemical challenges of chelating this large tetravalent f-block cation are considerable. Using the CD20-targeting antibody ofatumumab, we evaluated chelation of 227Th4+ for α-particle-emitting and radiotheranostic applications. Methods: We compared 4 bifunctional chelators for thorium radiopharmaceutical preparation: S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2-(4-isothicyanatobenzyl)-1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid (p-SCN-Bn-HEHA), p-isothiacyanatophenyl-1-hydroxy-2-oxopiperidine-desferrioxamine (DFOcyclo*-p-Phe-NCS), and macrocyclic 1,2-HOPO N-hydroxysuccinimide (L804-NHS). Immunoconstructs were evaluated for yield, purity, and stability in vitro and in vivo. Tumor targeting of the lead 227Th-labeled compound in vivo was performed in CD20-expressing models and compared with a companion 89Zr-labeled PET agent. Results: 227Th-labeled ofatumumab-chelator constructs were synthesized to a radiochemical purity of more than 95%, excepting HEHA. 227Th-HEHA-ofatumumab showed moderate in vitro stability. 227Th-DFOcyclo*-ofatumumab presented excellent 227Th labeling efficiency; however, high liver and spleen uptake was revealed in vivo, indicative of aggregation. 227Th-DOTA-ofatumumab labeled poorly, yielding no more than 5%, with low specific activity (0.08 GBq/g) and modest long-term in vitro stability (<80%). 227Th-L804-ofatumumab coordinated 227Th rapidly and efficiently at high yields, purity, and specific activity (8 GBq/g) and demonstrated extended stability. In vivo tumor targeting confirmed the utility of this chelator, and the diagnostic analog, 89Zr-L804-ofatumumab, showed organ distribution matching that of 227Th to delineate SU-DHL-6 tumors. Conclusion: Commercially available and novel chelators for 227Th showed a range of performances. The L804 chelator can be used with potent radiotheranostic capabilities for 89Zr/227Th quantitative imaging and α-particle therapy.

Aberrant centrosome biogenesis disrupts nephron progenitor cell renewal and fate resulting in fibrocystic kidney disease.

Mutations that disrupt centrosome structure or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet, it remains unclear how mutations in proteins essential for centrosome biogenesis impact embryonic kidney development. Here, we examined the consequences of conditional deletion of a ciliopathy gene, Cep120 , in the two nephron progenitor niches of the embryonic kidney. Cep120 loss led to reduced abundance of both metanephric mesenchyme and ureteric bud progenitor populations. This was due to a combination of delayed mitosis, increased apoptosis, and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis, and decline in filtration function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in pathways essential for branching morphogenesis, cystogenesis and fibrosis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney development, and identifies new therapeutic targets for renal centrosomopathies. Highlights: Defective centrosome biogenesis in nephron progenitors causes:Reduced abundance of metanephric mesenchyme and premature differentiation into tubular structuresAbnormal branching morphogenesis leading to reduced nephron endowment and smaller kidneysChanges in cell-autonomous and paracrine signaling that drive cystogenesis and fibrosisUnique cellular and developmental defects when compared to Pkd1 knockout models.

Cure of Disseminated Human Lymphoma with [225Ac]Ac-Ofatumumab in a Preclinical Model.

Immunotherapies that target the CD20 protein expressed on most non-Hodgkin lymphoma cells have improved clinical outcomes, but relapse is common. We prepared 225Ac-labeled anti-CD20 ofatumumab and evaluated its in vitro characteristics and therapeutic efficacy in a murine model of disseminated human lymphoma. Methods: 225Ac was chelated by DOTA-ofatumumab, and radiochemical yield, purity, immunoreactivity, stability, and chelate number were determined. In vitro cell killing of CD20-positive, human B-cell lymphoma Raji-Luc cells was assayed. Biodistribution was determined as percentage injected activity per gram (%IA/g) in mice with subcutaneous Raji-cell tumors (n = 4). [225Ac]Ac-ofatumumab biodistribution in C57BL/6N mice was performed to estimate projected human dosimetry. Therapeutic efficacy was tested in mice with systemically disseminated Raji-Luc cells, tracking survival, bioluminescence, and animal weight for a targeted 200 d, with single-dose therapy initiated 8, 12, or 16 d after cell injection, comparing no treatment, ofatumumab, and low (3.7 kBq/mouse) and high (9.25 kBq/mouse) doses of [225Ac]Ac-IgG and [225Ac]Ac-ofatumumab (n = 8-10/cohort). Results: Radiochemical yield and purity were 32% ± 9% and more than 95%, respectively. Specific activity was more than 5 MBq/mg. Immunoreactivity was preserved, and more than 90% of the 225Ac remained chelated after 10 d in serum. Raji-Luc cell killing in vitro was significant, specific, and dose-dependent. In tumor-bearing mice, [225Ac]Ac-ofatumumab displayed low liver (7 %IA/g) and high tumor (28 %IA/g) uptake. Dosimetry estimates indicated that bone marrow is likely the dose-limiting organ. When therapy was initiated 8 d after cell injection, untreated mice and mice treated with cold ofatumumab or low- or high-dose [225Ac]Ac-IgG showed indistinguishable median survivals of 20-24 d, with extensive cancer-cell burden before death. Low- and high-dose [225Ac]Ac-ofatumumab profoundly (P < 0.05) extended median survival to 190 d and more than 200 d (median not determinable), with 5 and 9 of 10 mice, respectively, surviving at study termination with no detectable cancer cells. Surviving mice treated with high-dose [225Ac]Ac-ofatumumab showed reduced weight gain versus naïve mice. When therapy was initiated 12 d, but not 16 d, after cell injection, high-dose [225Ac]Ac-ofatumumab significantly extended median survival to 40 d but was not curative. Conclusion: In an aggressive disseminated tumor model, [225Ac]Ac-ofatumumab was effective at cancer-cell killing and curative when administered 8 d after cell injection. [225Ac]Ac-ofatumumab has substantial potential for clinical translation as a next-generation therapeutic for treatment of patients with non-Hodgkin lymphoma.

Cure of Disseminated Human Lymphoma with [177Lu]Lu-Ofatumumab in a Preclinical Model.

Although immunotherapies that target CD20 on most non-Hodgkin lymphoma (NHL) cells have improved patient outcomes, current therapies are inadequate because many cases are, or become, refractory or undergo relapse. Here, we labelled the third-generation human anti-CD20 antibody ofatumumab with 177Lu, determined the in vitro characteristics of [177Lu]Lu-ofatumumab, estimated human dosimetry, and assayed tumor targeting and therapeutic efficacy in a murine model of disseminated NHL. Methods: CHX-A″-diethylenetriaminepentaacetic acid-[177Lu]Lu-ofatumumab was prepared. We evaluated radiochemical yield, purity, in vitro immunoreactivity, stability, (n = 7), affinity, and killing of CD20-expressing Raji cells (n = 3). Human dosimetry was estimated from biodistribution studies as percentage injected activity per gram using C57BL/6N mice. Tissue and organ biodistribution was determined in R2G2 immunodeficient mice with subcutaneous Raji-cell tumors. Therapy studies used R2G2 mice with disseminated human Raji-luc tumor cells (n = 10 mice/group). Four days after cell injection, the mice were left untreated or were treated with ofatumumab, 8.51 MBq of [177Lu]Lu-IgG, or 0.74 or 8.51 MBq of [177Lu]Lu-ofatumumab. Survival, weight, and bioluminescence were tracked. Results: Radiochemical yield was 93% ± 2%, radiochemical purity was 99% ± 1%, and specific activity was 401 ± 17 MBq/mg. Immunoreactivity was substantially preserved, and more than 75% of 177Lu remained chelated after 7 d in serum. [177Lu]Lu-ofatumumab specifically killed Raji-luc cells in vitro (P < 0.05). Dosimetry estimated that an effective dose for human administration is 0.36 mSv/MBq and that marrow may be the dose-limiting organ. Biodistribution in subcutaneous tumors 1, 3, and 7 d after [177Lu]Lu-ofatumumab injection was 11, 15, and 14 percentage injected activity per gram, respectively. In the therapy study, median survival of untreated mice was 19 d, not statistically different from mice treated with 8.51 MBq of [177Lu]Lu-IgG (25 d). Unlabeled ofatumumab increased survival to 46 d, similar to 0.74 MBq of [177Lu]Lu-ofatumumab (59 d), with both being superior to no treatment (P < 0.0003). Weight loss and increased tumor burden preceded death or killing of the animal for cause. In contrast, treatment with 8.51 MBq of [177Lu]Lu-ofatumumab dramatically increased median survival (>221 d), permitted weight gain, eliminated detectable tumors, and was curative in 9 of 10 mice. Conclusion: [177Lu]Lu-ofatumumab shows favorable in vitro characteristics, localizes to tumor, and demonstrates curative therapeutic efficacy in a disseminated lymphoma model, showing potential for clinical translation to treat NHL.

A Possible Pathogenic PSEN2 Gly56Ser Mutation in a Korean Patient with Early-Onset Alzheimer's Disease.

Early-onset Alzheimer's disease (EOAD) is characterized by the presence of neurological symptoms in patients with Alzheimer's disease (AD) before 65 years of age. Mutations in pathological genes, including amyloid protein precursor (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2), were associated with EOAD. Seventy-six mutations in PSEN2 have been found around the world, which could affect the activity of γ-secretase in amyloid beta processing. Here, a heterozygous PSEN2 point mutation from G to A nucleotide change at position 166 (codon 56; c.166G>A, Gly56Ser) was identified in a 64-year-old Korean female with AD with progressive cognitive memory impairment for the 4 years prior to the hospital visit. Hippocampal atrophy was observed from magnetic resonance imaging-based neuroimaging analyses. Temporal and parietal cortex hypometabolisms were identified using fluorodeoxyglucose positron emission tomography. This mutation was at the N-terminal portion of the presenilin 2 protein on the cytosolic side. Therefore, the serine substitution may have promoted AD pathogenesis by perturbing to the mutation region through altered phosphorylation of presenilin. In silico analysis revealed that the mutation altered protein bulkiness with increased hydrophilicity and reduced flexibility of the mutated region of the protein. Structural changes were likely caused by intramolecular interactions between serine and other residues, which may have affected APP processing. The functional study will clarify the pathogenicity of the mutation in the future.

Cilioretinal Artery Occlusion after Endovascular Coil Embolization for Anterior Communicating Artery.

Unruptured intracranial aneurysms have a risk of rupture, so coil embolization is widely practiced as it preserves a patent artery. There are complications of coil procedures, such as patent artery occlusion and thromboembolism, which can result in retinal artery occlusion. We report onretinal artery occlusion following coil embolization of anterior communicating artery aneurysm. This is a rare case of a combination of cilioretinal and branch retinal artery occlusion, and is unusual in showing a functional recovery.

Bioluminescent Tumor Signal Is Mouse Strain and Pelt Color Dependent: Experience in a Disseminated Lymphoma Model.

BACKGROUND: Many preclinical cancer studies use mice with varied phenotypes to monitor tumor treatment. We compared survival and optical imaging characteristics of strains with varied coat colors harboring luciferase-expressing disseminated lymphoma. RESULTS: Luciferase-expressing lymphoma cells (Raji-luc) were injected via tail vein into severe combined immunodeficient (SCID) and Rag2-IL2rg (R2G2) mice, and survival was tracked. Tumor signals were obtained by imaging ventral and dorsal aspects of mice. Signal attenuation by isolated mouse pelts was measured in vitro. R2G2 mice had decreased survival compared to SCID mice (17 vs. 32 days, p<0.001) despite similar bioluminescence signal when mice were imaged dorsally (p=0.37). However, signal was 17.3-fold higher in R2G2 mice compared to SCID (p<0.001) when imaged ventrally. Isolated dark R2G2 dorsal pelts attenuated signal more than ventral pelts when placed over cells in vitro. CONCLUSIONS: Mouse pelt color and imaging aspect are critical considerations for quantifying bioluminescent tumor signal, and the R2G2 mouse strain may prove useful for preclinical targeted therapy studies.

Multimodal Mesoporous Silica Nanocarriers for Dual Stimuli-Responsive Drug Release and Excellent Photothermal Ablation of Cancer Cells.

BACKGROUND: Core-shell types of mesoporous silica nanoparticles (MSNs) with multimodal functionalities were developed for bio-imaging, controlled drug release associated with external pH, and near-infrared radiation (NIR) stimuli, and targeted and effective chemo-photothermal therapeutics. MATERIALS AND METHODS: We synthesized and developed a core-shell type of mesoporous silica nanocarriers for fluorescent imaging, stimuli-responsive drug release, magnetic separation, antibody targeting, and chemo-photothermal therapeutics. Also, the biocompatibility, cellular uptake, cytotoxicity, and photothermal therapy on these FS3-based nanocarriers were systematically investigated. RESULTS: Magnetic mesoporous silica nanoparticles was prepared by coating a Fe3O4 core with a mesoporous silica shell, followed by grafting with fluorescent conjugates, so-called FS3. The resulting FM3 was preloaded with therapeutic cisplatin and coated with polydopamine layer, so-called FS3P/C. Eventually, graphene oxide-wrapped FS3P/C (FS3P-G/C) exhibited high sensitivity in the dual stimuli (pH, NIR)-responsive controlled release behavior. On the other hand, Au NPs-coated FS3P/C (FS3P-A/C) exhibited more stable release behavior, irrespective of pH changes, and exhibited much more enhanced release rate under the same NIR irradiation. Notably, FS3P-A/C showed strong NIR absorption, enabling photothermal destruction of HeLa cells by its chemo-photothermal therapeutic effects under NIR irradiation (808 nm, 1.5 W/cm2). The selective uptake of FS3-based nanocarriers was confirmed in cancer cell lines including HeLa (American Type Culture Collection - ATCC) and SHSY5Y (ATCC 2266) by the images obtained from confocal laser scanning microscopy, flow cytometry, and transmission electron microscopy instruments. Cisplatin-free FS3-based nanocarriers revealed good cellular uptake and low cytotoxicity against cancerous HeLa and SH-SY5Y cells, but showed no obvious toxicity to normal HEK293 (ATCC 1573) cell. CONCLUSION: Along with the facile synthesis of FS3-based nanocarriers, the integration of all these strategies into one single unit will be a prospective candidate for biomedical applications, especially in chemo-photothermal therapeutics, targeted delivery, and stimuli-responsive controlled drug release against multiple cancer cell types.

Additional diagnostic testing of the 2019 novel coronavirus (SARS-CoV-2).

PURPOSE OF REVIEW: Within the last two decades several members of the Coronaviridae family namely Severe Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome (MERS-CoV) have demonstrated epidemic potential. In late, 2019 an unnamed genetic relative, later named SARS-CoV-2 realized its potential in the highly populous neighborhoods of Wuhan, China. Unchecked, the virus rapidly spread among interconnected communities and related households before containment measures could be in acted. "Appropriate" diagnostic testing in response to the SARS-CoV-2 outbreak should be urgently considered. This perspective review gives particular attention to the potential diagnostic testing of the virus in semen and seminal fluids due to its high levels of angiotensin converting enzyme 2 (ACE2) precursor. RECENT FINDINGS: As many infectious viruses are stable in semen and have transmitted the respective diseases, the presence of SARS-CoV-2 should be tested in semen to assess their stabilities and half-life. As in case of Ebola virus, it was present in semen for longer period in a carrier man without any symptom. Additional hypothesis is that since ACE2 could serve as a mediator for the endocytosis of the previously SARS coronavirus, SARS-CoV-2 may enter the cells through similar mechanism. From the protein expression atlas, high levels of ACE2 precursor were found in intestines and testis. Hence, the testis and seminal fluids could be the host cell and/or reservoir. The results could be used as a suggestive guideline for the sexual activities after the discharge or declaration of disease free.

Novel amyloid precursor protein mutation, Val669Leu ("Seoul APP"), in a Korean patient with early-onset Alzheimer's disease.

In this study, a novel mutation in APP gene, Val669Leu ("Seoul APP"), was reported in a Korean female patient with Alzheimer's disease. She developed cognitive decline at 56 years of age, and her memory declined rapidly over one-year period from her 1st visit to the hospital. Her Mini-Mental State Examination scores dropped from 25/30 to 13/30. Two years later, she developed parkinsonian features, myoclonic jerk, and generalized seizure. As the disease progressed, aggravated diffuse brain atrophy and small-vessel ischemic lesion was also observed, and she became mute and vegetative in 4 years from the symptom onset. Magnetic resonance imaging showed mild medial temporal lobe and hippocampal atrophy, and 18F-fluoro-deoxyglucose positron emission tomography showed bilateral temporoparietal hypometabolism. Plasma amyloid oligomer analysis revealed highly elevated Aß oligomers levels in the proband patient. Family history revealed positive without biochemical confirmation because family members testified similar type of cognitive decline from the proband's mother and one of her aunt/uncle. Her half-siblings did not present any signs of memory impairment. Sanger sequencing of the proband patient revealed a novel mutation in APP gene, Val669Leu, but mutation was not found in her unaffected half-sisters. A designed algorithm by Guerreiro et al. on early-onset Alzheimer's disease-associated mutations suggested the mutation as possibly pathogenic mutation. On the other hand, PolyPhen2 and SIFT tools suggested as otherwise. Since the mutation was located nearby the ß-secretase cleavage site of APP, right next to the Swedish APP (Lys,Met670/671Asn,Leu) mutation, it was named as "Seoul APP" mutation. 3D modeling revealed that this mutation could result in significant changes in loop orientation of APP and also its intramolecular interactions. Hence, a novel APP Val669Leu mutation could alter the binding interactions between APP and ß-secretase, which may influence the Aß40 and Aß42 generations.

Influenza mimetic protein-polymer nanoparticles as antigen delivery vehicles to dendritic cells for cancer immunotherapy.

Stimulation of dendritic cells (DCs) by antigens (Ags) promotes an Ag-specific immune response that kills Ag-expressing pathogens. These biologically inspired nanocarriers have received much attention as tools to deliver cancer Ags to DCs. A polymer-templated protein nanoball having hemagglutinin (H1-NB) that mimics the influenza virus can be used as a cancer Ag delivery vehicle, as DCs show effective phagocytic activities against H1-NB without any adjuvant. In the present study, H1-NB containing ovalbumin (OVA), a model Ag (H1-OVA-NB), was prepared as an anti-cancer agent and evaluated for its effect on anticancer immunity. H1-OVA-NB treatments in C57BL/6 mice enhanced OVA-specific immune activation and efficiently inhibited B16-OVA tumor growth compared to control groups. Our results indicate that H1-NB is an effective carrier for Ag delivery to DCs and promotes immunotherapy to fight cancer.

Regulation of cilia abundance in multiciliated cells.

Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells.

Arginine reprogramming in ADPKD results in arginine-dependent cystogenesis.

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

Virus-mimetic polymer nanoparticles displaying hemagglutinin as an adjuvant-free influenza vaccine.

The generation of virus-mimetic nanoparticles has received much attention in developing a new vaccine for overcoming the limitations of current vaccines. Thus, a method, encompassing most viral features for their size, hydrophobic domain and antigen display, would represent a meaningful direction for the vaccine development. In the present study, a polymer-templated protein nanoball with direction oriented hemagglutinin1 on its surface (H1-NB) was prepared as a new influenza vaccine, exhibiting most of the viral features. Moreover, the concentrations of antigen on the particle surface were controlled, and its effect on immunogenicity was estimated by in vivo studies. Finally, H1-NB efficiently promoted H1-specific immune activation and cross-protective activities, which consequently prevented H1N1 infections in mice.

Orientation Controlled Protein Nanocapsules by Enzymatic Removal of a Polymer Template.

Protein nanocapsules are potentially useful as functional nanocarriers because of their hollow structure and high biocompatibility and the intrinsic activity of their protein constituents. However, the development of a facile method for the preparation of oriented nanocapsules that retain their protein activity has been challenging. Here we describe the preparation of protein nanocapsules through the enzymatic removal of polymer templates. Nickel(II) nitrilotriacetic acid-end-functionalized poly(lactic acid) (Ni2+-NTA-PLA) was introduced as a polymeric template to immobilize hexa-histidine-tagged green fluorescence protein (His6-GFP) with consistent orientation. Following protein cross-linking and core-degradation, various measurements as a function of degradation time indicated the formation of hollow structures. We also demonstrated orientational control and activity preservation of the protein after capsule preparation. Protein nanocapsules prepared by this method can act as functional containers, taking advantage of the intrinsic function of their constituent proteins without additional modification.

Centrosome amplification disrupts renal development and causes cystogenesis.

Centrosome number is tightly controlled to ensure proper ciliogenesis, mitotic spindle assembly, and cellular homeostasis. Centrosome amplification (the formation of excess centrosomes) has been noted in renal cells of patients and animal models of various types of cystic kidney disease. Whether this defect plays a causal role in cystogenesis remains unknown. Here, we investigate the consequences of centrosome amplification during kidney development, homeostasis, and after injury. Increasing centrosome number in vivo perturbed proliferation and differentiation of renal progenitors, resulting in defective branching morphogenesis and renal hypoplasia. Centrosome amplification disrupted mitotic spindle morphology, ciliary assembly, and signaling pathways essential for the function of renal progenitors, highlighting the mechanisms underlying the developmental defects. Importantly, centrosome amplification was sufficient to induce rapid cystogenesis shortly after birth. Finally, we discovered that centrosome amplification sensitized kidneys in adult mice, causing cystogenesis after ischemic renal injury. Our study defines a new mechanism underlying the pathogenesis of renal cystogenesis, and identifies a potentially new cellular target for therapy.

Ambient-temperature co-digestion of low-solids municipal and industrial waste mixtures: Insights from molecular analyses.

The performance of ambient temperature anaerobic co-digestion was investigated for mixtures of six substrates: canned tomato and salsa waste, portable toilet waste, septic tank waste, winery waste, beer and cider waste, and fats, oils, and grease (FOG). Laboratory semi-continuous reactor studies and molecular biological analyses revealed that beer/winery, and tomato/FOG/winery/beer mixtures resulted in the best performance in terms of biogas production (515 and 371 mL CH4/g VS, respectively) and methanogenic populations. A portable toilet/septage mixture resulted in the overall poorest performance and inhibition of microbial activity was evident. Average methane content was ~70% for all mixtures tested. The findings of this study reveal that healthy methanogen populations were present, further supporting the feasibility of biogas production via the novel feedstock mixtures in ambient temperature lagoons. Implications: Disposal of septic tank waste and other high chemical oxygen demand (COD) 10 industrial food processing waste at a small wastewater treatment plant is uncommon, because it can upset the treatment process and requires additional power for treatment. Ambient-temperature covered lagoon digesters can be an alternative low-cost technology for co-digestion of these recalcitrant waste streams while generating bioenergy. The results of this study demonstrated that there is potential for implementation of unheated covered lagoon digester systems 15 for conversion of liquid wastes for production of renewable biomethane while eliminating the need to treat these wastes at a wastewater treatment plant.

Targeted and controlled drug delivery by multifunctional mesoporous silica nanoparticles with internal fluorescent conjugates and external polydopamine and graphene oxide layers.

This study demonstrated the targeted delivery and controlled release of cisplatin drug molecules from doubly decorated mesoporous silica nanoparticles (MSNs), which were internally grafted with fluorescent conjugates and externally coated with polydopamine (PDA) and graphene oxide (GO) layers. The brush-like internal conjugates conferred fluorescent functionality and high capacity of cisplatin loading into MSNs, as well as contributing to a sustained release of the cisplatin through a porous channel with the assistance of external PDA layer. A consolidated double-layer formed by electrostatic interactions between the GO nanosheet and the PDA layer induced more controlled release kinetics which was well predicted by Higuchi model. In addition, Our MSNs exhibited stimuli (pH, NIR irradiation)-responsive controlled release as a potential chemo-photothermal agent against cancer cells. In a cell test, multifunctional MSNs showed a low toxicity itself, but gave a high cytotoxicity against human epithelial neuroblastoma cells (SH-SY5Y) after loading cisplatin. Notably, GO-wrapped MSNs exhibited very effective drug delivery because GO wrapping enhanced their dispensability in aqueous solution, photothermal heating effect, and efficient endocytosis into cells. Furthermore, monoclonal antibody (anti-human epidermal growth factor receptor)-conjugated MSNs showed a higher specificity, which resulted in more enhanced anticancer effects in vitro. The current study demonstrated a reliable synthesis of multifunctional MSNs, endowed with fluorescent imaging, stimuli-responsive controlled release, higher specificity, and efficient cytotoxicity toward cancer cells. STATEMENT OF SIGNIFICANCE: The current study demonstrated the reliable synthesis of multifunctional mesoporous silica nanoparticles (MSNs) with internal fluorescent conjugates and external polydopamine and graphene oxide (GO) layers. The combination of internal conjugates and external coating layers produced an effective pore closure effect, leading to controlled and sustained release of small drug molecules. Notably, GO wrapping improved the dispensability and cellular uptake of the MSNs, as well as enhanced drug-controlled release. Our multifunctional MSNs revealed very efficient drug delivery effects against human epithelial neuroblastoma cells by demonstrating several strengths: i) fluorescent imaging, ii) sustained and controlled release of small drug molecules, iii) efficient cellular uptake, cytotoxicity and specificity, and v) stimuli (pH, NIR irradiation)-responsive controlled release as a potential chemo-photothermal agent.