Anders Kvist

MiRNA expression in urothelial carcinomas: important roles of miR-10a, miR-222, miR-125b, miR-7 and miR-452 for tumor stage and metastasis, and frequent homozygous losses of miR-31.

We analyzed 34 cases of urothelial carcinomas by miRNA, mRNA and genomic profiling. Unsupervised hierarchical clustering using expression information for 300 miRNAs produced 3 major clusters of tumors corresponding to Ta, T1 and T2‐T3 tumors, respectively. A subsequent SAM analysis identified 51 miRNAs that discriminated the 3 pathological subtypes. A score based on the expression levels of the 51 miRNAs, identified muscle invasive tumors with high precision and sensitivity. MiRNAs showing high expression in muscle invasive tumors included miR‐222 and miR‐125b and in Ta tumors miR‐10a. A miRNA signature for FGFR3 mutated cases was also identified with miR‐7 as an important member. MiR‐31, located in 9p21, was found to be homozygously deleted in 3 cases and miR‐452 and miR‐452* were shown to be over expressed in node positive tumors. In addition, these latter miRNAs were shown to be excellent prognostic markers for death by disease as outcome. The presented data shows that pathological subtypes of urothelial carcinoma show distinct miRNA gene expression signatures.

Carrying large fuel loads during sustained bird flight is cheaper than expected

Birds on migration alternate between consuming fuel stores during flights and accumulating fuel stores during stopovers. The optimal timing and length of flights and stopovers for successful migration depend heavily on the extra metabolic power input (fuel use) required to carry the fuel stores during flight. The effect of large fuel loads on metabolic power input has never been empirically determined. We measured the total metabolic power input of a long-distance migrant, the red knot (Calidris canutus), flying for 6 to 10 h in a wind tunnel, using the doubly labelled water technique. Here we show that total metabolic power input increased with fuel load, but proportionally less than the predicted mechanical power output from the flight muscles. The most likely explanation is that the efficiency with which metabolic power input is converted into mechanical output by the flight muscles increases with fuel load. This will influence current models of bird flight and bird migration. It may also help to explain why some shorebirds, despite the high metabolic power input required to fly, routinely make nonstop flights of 4,000 km longer.

Identification of new microRNAs in paired normal and tumor breast tissue suggests a dual role for the ERBB2/Her2 gene.

To comprehensively characterize microRNA (miRNA) expression in breast cancer, we performed the first extensive next-generation sequencing expression analysis of this disease. We sequenced small RNA from tumors with paired samples of normal and tumor-adjacent breast tissue. Our results indicate that tumor identity is achieved mainly by variation in the expression levels of a common set of miRNAs rather than by tissue-specific expression. We also report 361 new, well-supported miRNA precursors. Nearly two-thirds of these new genes were detected in other human tissues and 49% of the miRNAs were found associated with Ago2 in MCF7 cells. Ten percent of the new miRNAs are located in regions with high-level genomic amplifications in breast cancer. A new miRNA is encoded within the ERBB2/Her2 gene and amplification of this gene leads to overexpression of the new miRNA, indicating that this potent oncogene and important clinical marker may have two different biological functions. In summary, our work substantially expands the number of known miRNAs and highlights the complexity of small RNA expression in breast cancer.

The non-coding RNA of the multidrug resistance-linked vault particle encodes multiple regulatory small RNAs.

Vault particles are conserved organelles implicated in multidrug resistance and intracellular transport. They contain three different proteins and non-coding vault RNAs (vRNAs). Here we show that human vRNAs produce several small RNAs (svRNAs) by mechanisms different from those in the canonical microRNA (miRNA) pathway. At least one of these svRNAs, svRNAb, associates with Argonaute proteins to guide sequence-specific cleavage and regulate gene expression similarly to miRNAs. We demonstrate that svRNAb downregulates CYP3A4, a key enzyme in drug metabolism. Our findings expand the repertoire of small regulatory RNAs and assign, for the first time, a function to vRNAs that may help explain the association between vault particles and drug resistance.

Inhibition of HIV-1 Disease Progression by Contemporaneous HIV-2 Infection

BACKGROUND Progressive immune dysfunction and the acquired immunodeficiency syndrome (AIDS) develop in most persons with untreated infection with human immunodeficiency virus type 1 (HIV-1) but in only approximately 20 to 30% of persons infected with HIV type 2 (HIV-2); among persons infected with both types, the natural history of disease progression is poorly understood. METHODS We analyzed data from 223 participants who were infected with HIV-1 after enrollment (with either HIV-1 infection alone or HIV-1 and HIV-2 infection) in a cohort with a long follow-up duration (approximately 20 years), according to whether HIV-2 infection occurred first, the time to the development of AIDS (time to AIDS), CD4+ and CD8+ T-cell counts, and measures of viral evolution. RESULTS The median time to AIDS was 104 months (95% confidence interval [CI], 75 to 133) in participants with dual infection and 68 months (95% CI, 60 to 76) in participants infected with HIV-1 only (P=0.003). CD4+ T-cell levels were higher and CD8+ T-cell levels increased at a lower rate among participants with dual infection, reflecting slower disease progression. Participants with dual infection with HIV-2 infection preceding HIV-1 infection had the longest time to AIDS and highest levels of CD4+ T-cell counts. HIV-1 genetic diversity was significantly lower in participants with dual infections than in those with HIV-1 infection alone at similar time points after infection. CONCLUSIONS Our results suggest that HIV-1 disease progression is inhibited by concomitant HIV-2 infection and that dual infection is associated with slower disease progression. The slower rate of disease progression was most evident in participants with dual infection in whom HIV-2 infection preceded HIV-1 infection. These findings could have implications for the development of HIV-1 vaccines and therapeutics. (Funded by the Swedish International Development Cooperation Agency-Swedish Agency for Research Cooperation with Developing Countries and others.).

MAXIMUM ENERGY INTAKE RATE IS PROPORTIONAL TO BASAL METABOLIC RATE IN PASSERINE BIRDS

A high energy intake rate may be important for many animals, but little is known of factors that determine intake capacity. Birds in migratory disposition presumably eat at their maximum capacity and therefore form a good model for studying the energy intake capacity of animals. We measured the maximum daily gross energy intake (GEImax) and basal metabolic rates (BMR) in 22 species of migrant passerines during autumn. Both GEImax and BMR scaled to body mass (w) close to w0.70. More importantly, species with high GEImax for their body mass also had relatively high BMR. BMR may reflect the size of the metabolic machinery involved in energy uptake. The maximum daily metabolizable energy intake (DMEmax) was estimated from our GEimax data. The average ratio DMEmax/ BMR was 4.6. In comparison, daily energy expenditure of reproducing passerines have been reported in the literature to be 3.6 times BMR. This suggests that energy intake rates may not normally limit breeding performance in passerines. Earlier studies have shown that BMR of homoeotherms reflect the rate of energy expenditure during reproduction. Our study shows that the energy intake capacity also correlates with BMR, which gives new perspectives on the ecological significance of BMR.

Maximum daily energy intake: it takes time to lift the metabolic ceiling.

Conventionally, maximum capacities for energy assimilation are presented as daily averages. However, maximum daily energy intake is determined by the maximum metabolizable energy intake rate and the time available for assimilation of food energy. Thrush nightingales (Luscinia luscinia) in migratory disposition were given limited food rations for 3 d to reduce their energy stores. Subsequently, groups of birds were fed ad lib. during fixed time periods varying between 7 and 23 h per day. Metabolizable energy intake rate, averaged over the available feeding time, was 1.9 W and showed no difference between groups on the first day of refueling. Total daily metabolizable energy intake increased linearly with available feeding time, and for the 23‐h group, it was well above suggested maximum levels for animals. We conclude that both intake rate and available feeding time must be taken into account when interpreting potential constraints acting on animals’ energy budgets. In the 7‐h group, energy intake rates increased from 1.9 W on the first day to 3.1 W on the seventh day. This supports the idea that small birds can adaptively increase their energy intake rates on a short timescale.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Differences in molecular evolution between switch (R5 to R5X4/X4-tropic) and non-switch (R5-tropic only) HIV-1 populations during infection.

The recent introduction of entry inhibitors in the clinic as components of antiretroviral treatment has heightened the interest in coreceptor use of human immunodeficiency virus type 1 (HIV-1). Viruses using CCR5 as coreceptor (R5 viruses) are generally present over the entire course of infection whereas viruses using the CXCR4 coreceptor (R5X4/X4 viruses) emerge in about 50% of infected individuals during later stages of infection. The CCR5-to-CXCR4 switch represents a concern because CCR5 inhibitors, while suppressing R5 viruses, may allow the emergence of CXCR4-tropic viruses. In this study, HIV-1 populations that maintained CCR5 usage during infection were compared with populations that switched coreceptor usage to include CXCR4 later during infection, with the aim to find molecular properties of the virus populations associated with the CCR5-to-CXCR4 switch. We amplified and molecularly cloned the V1-V3 region of the HIV-1 envelope from 51 sequential HIV-1 isolates derived from 4 to 10 serial samples for each of the patients. Four of the patients had virus populations that switched coreceptor usage to include CXCR4 (switch populations: SP) during infection and four patients had viral populations that maintained exclusive CCR5 usage (non-switch populations: nSP). Coreceptor usage was determined experimentally on individual clones from dualtropic R5X4 isolates. In nSP we found that the number of potential N-linked glycosylation sites (PNGS) increased over time, whereas no pattern of change was observed in SP. We also found differences in V2 length and V3 charge between R5 viruses of nSP and R5 viruses of SP before the switch. The V2 region was significantly longer in R5 viruses of SP compared to viruses of nSP throughout the course of infection, and the V3 charge increased with time in R5 populations from SP, while it remained unchanged or decreased in nSP. These molecular properties could prove important for understanding the evolution of coreceptor usage in HIV-1 populations, and maybe even for predicting an upcoming coreceptor switch at early stages after primary infection.

Multiregion whole-exome sequencing uncovers the genetic evolution and mutational heterogeneity of early-stage metastatic melanoma

Cancer genome sequencing has shed light on the underlying genetic aberrations that drive tumorigenesis. However, current sequencing-based strategies, which focus on a single tumor biopsy, fail to take into account intratumoral heterogeneity. To address this challenge and elucidate the evolutionary history of melanoma, we performed whole-exome and transcriptome sequencing of 41 multiple melanoma biopsies from eight individual tumors. This approach revealed heterogeneous somatic mutations in the range of 3%-38% in individual tumors. Known mutations in melanoma drivers BRAF and NRAS were always ubiquitous events. Using RNA sequencing, we found that the majority of mutations were not expressed or were expressed at very low levels, and preferential expression of a particular mutated allele did not occur frequently. In addition, we found that the proportion of ultraviolet B (UVB) radiation-induced C>T transitions differed significantly (P < 0.001) between early and late mutation acquisition, suggesting that different mutational processes operate during the evolution of metastatic melanoma. Finally, clinical history reports revealed that patients harboring a high degree of mutational heterogeneity were associated with more aggressive disease progression. In conclusion, our multiregion tumor-sequencing approach highlights the genetic evolution and non-UVB mutational signatures associated with melanoma development and progression, and may provide a more comprehensive perspective of patient outcome. Cancer Res; 76(16); 4765-74. ©2016 AACR.