Considerations for analysis of time-to-event outcomes subject to competing risks in veterinary clinical studies

In veterinary medicine, prospective clinical trials are increasingly utilized to address questions regarding effectiveness of therapies and patient prognosis. A large number of these trials involve time-to-event (TTE) endpoints, which require special methods of analysis to handle data in which not all subjects are observed to have the event of interest. Analyses and interpretation of the results can be further complicated when an endpoint of interest is not observed in some patients because they incur a competing risk, such as death from an unrelated cause. Competing risks have been the source of confusion in many epidemiologic analyses leading to the potential for misinterpretation. In this article, we review key considerations for the TTE analysis in the setting of competing risks. We briefly review standard TTE tools, namely Kaplan–Meier survival curves and Cox regression. In the setting of outcomes with competing risks, we provide guidance on the appropriate analysis techniques, such as cumulative incidence curves, to estimate the risk of an event of interest. We also describe a common pitfall of treating competing risks as censoring in Kaplan–Meier survival curve analysis, which can overestimate the event rate of interest. We describe two common regression methods that examine associated risk factors in the presence of competing risks and highlight the different research questions these methods address. This article provides an introductory overview and illustrates concepts with examples from veterinary trials and with example data sets.     

Investigation of ventricular pre-excitation electrocardiographic pattern in two horses: clinical presentation and potential causes

Two horses referred to the Unitat Equina, Fundació Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, for unrelated clinical problems, and with no previous history of cardiac disease exhibited an intermittent ventricular pre-excitation electrocardiographic pattern during hospitalization. Both animals showed decreased plasma total and ionized magnesium concentrations, but no other relevant electrolyte disturbances were detected. Altered interventricular septal motion associated with ventricular pre-excitation beats (VPBs) was detected on M-mode echocardiography in both horses. The likely localization of an accessory pathway (AP) was identified in case 2 using pulsed-wave tissue Doppler imaging in the left anterior paraseptal location. Decreased frequency of the VPB was observed with long-term magnesium supplementation and restoration of plasma magnesium concentrations. The presence of ventricular pre-excitation electrocardiographic pattern was attributed to higher sensitivity of the AP to hypomagnesemia in both cases.     

Population genome of the newly discovered Jinchuan yak to understand its adaptive evolution in extreme environments and generation mechanism of the multirib trait

The adaptation and diversity of animals to the extreme environments of the Qinghai–Tibet Plateau (QTP) are typical materials to study adaptive evolution. The recently discovered Jinchuan yak population has many individuals with multiple ribs. However, little is known about this yak's origin, evolution, and the genetic mechanisms that formed its unique multirib trait. Here, we report a valuable population genome resource of the Jinchuan yak by resequencing the whole genome of 150 individuals. Population genetic polymorphism and structure analysis reveal that Jinchuan yak can be differentiated as a unique and original yak population among the domestic yak. Combined with geological change, the Jinchuan yak's evolutionary origin is speculated to be about 6290 years ago, which may be related to the unique geographical environment of the eastern edge of the QTP during this period. Compared with other domestic yaks, this new population has 280 positively selected genes. The genes related to skeletal function hold a considerable and remarkable proportion, suggesting that the specific skeletal characteristics have been enhanced in the adaptive evolution of Jinchuan yak in the extreme plateau environment. The genome-wide association study has revealed that TUBA8 and TUBA4A, the genes that regulate the cytoskeleton, are potential genes associated with the multirib trait. Our findings provide a basis to further understand the generation mechanism of the adaptive evolution of this new population in high-altitude extreme environments and the multivertebrate trait of domestic animals.     

Prolonged drought changes the bacterial growth response to rewetting

Rewetting a dry soil can result in two response patterns of bacterial growth and respiration. In type 1, bacterial growth starts to increase linearly immediately upon rewetting and respiration rates are highest immediately upon rewetting. In type 2, bacterial growth starts to increase exponentially after a lag period with a secondary increase in respiration occurring at the start of the exponential increase in growth. We previously observed that the type 1 response occurred after rewetting 4-day dried soil and type 2 for 1-year dried soil. Here we studied in detail how the duration of drought related to the two types of responses of bacterial growth and respiration to rewetting. Soil was air dried for different time periods from 4 days up to 48 weeks. Upon rewetting, bacterial growth and respiration was measured repeatedly at 17 °C during one week. Drought periods of ≤2 weeks resulted in a type 1 response whereas drought periods of ≥4 weeks resulted in a type 2 response. The lag period increased with drought duration and reached a maximum of ca. 18 h. The bacterial growth response was also affected by incubation of moist soil before drying–rewetting. The lag period increased with duration of moist soil incubation before the 4-day drying–rewetting event and reached also a maximum of ca. 18 h. The exponential growth increase in the type 2 response coincided with a secondary increase in respiration, which increased in magnitude with increasing drought duration. Cumulative respiration increased with drought duration and was ca. 4 times higher after 48 weeks of drought compared to 4 days. Thus, prolonged drought affected the response type of bacterial growth and respiration to rewetting, and also increased lag period, the magnitude of the secondary increase in respiration and total C release. The effect of drought was, however, modified by the lenght of the incubation period of moist soil before drought, suggesting that soil conditions before a drying–rewetting event need consideration when evaluating microbial responses.     

Growth-dependent survival mechanisms during the early life of a temperate seabass (Lateolabrax japonicus): field test of the ‘growth–mortality’ hypothesis

Three concepts based on size, time and growth rate are contained in the functional mechanisms of the ‘growth–mortality’ hypothesis, and are known as the ‘bigger is better’, ‘stage duration’ and ‘growth-selective predation’ hypotheses, respectively. Although it is sufficiently documented in fishes that faster growing and bigger larvae selectively survive, synergistic operation of the three components of the ‘growth–mortality’ hypothesis within a species have received little attention. In this study, we have tested the components of the ‘growth–mortality’ hypothesis based on the growth characteristics of original population (OP) and survivors (SV) of Japanese seabass (JSB) Lateolabrax japonicus. Larval and juvenile JSB were repeatedly sampled from the Tango Sea in 2007 and 2008. Otoliths from larvae and juveniles were analyzed to produce back-calculated daily records of size-at-age and growth rate, and were compared between OP and SV. Selective survival for fast-growing and bigger larvae was evident, and our results strongly support the ‘bigger is better’ hypothesis as well as the ‘growth-selective predation’ hypothesis. Growth rates of the individuals that metamorphosed into juveniles had significantly faster growth rates than non-metamorphosing larvae older than 48 days, the minimum age for metamorphosis, suggesting a clear relationship between growth rates and the timing of metamorphosis. On average, SV completed the larval stage ∼13 days earlier than those of OP, supporting the ‘stage duration’ hypothesis. Thus, SV of JSB exhibited traits consistent with all aspects of the ‘growth–mortality’ hypothesis: faster growth, bigger size-at-age, and shorter larval stage duration (LSD), i.e., larvae with faster growth, bigger size-at-age and a shorter LSD selectively survived the larval period. Although maternal influence on growth and survival was evident, factors that regulate growth–mortality mechanisms remain to be explored for JSB.     

Carbon availability regulates soil respiration response to nitrogen and temperature

The response of soil CO₂ fluxes (R_soil) to interactions between carbon (C) and nitrogen (N) availability or C and temperature conditions is not well understood, but may increasingly affect future C storage under the combined anthropogenic impacts of N deposition and climate change. Here we addressed this uncertainty through a series of laboratory incubation experiments using soils from three contrasting ecosystems to investigate how changes in C, N, and temperature regulate R_soil through changes to Michaelis–Menten parameters (i.e. V_max and K_m). Results of this study demonstrate that R_soil response to N enrichment and changes in temperature are dependent on the C availability of soil substrates. N addition influenced R_soil through both the maximum rate (V_max) and the half saturation constant (K_m). The increase in K_m corresponded to a decrease in R_soil when C was limited. Alternatively, when C was abundant, N enrichment increased R_soil, which corresponded to an increase in V_max. Regulation of temperature sensitivity through V_max and K_m was also dependent on C availability. Both V_max and K_m demonstrated positive temperature responses, supporting the hypothesis of a canceling effect at low C concentrations. While temperature sensitivity was influenced by both C quantity and C complexity, our results suggested that C quantity is a stronger predictor. Despite strong differences in climate, vegetation, and management of our soils, C–N and C-temperature interactions were markedly similar between sites, highlighting the importance of C availability in the regulation of R_soil and justifying the use of Michaelis–Menten kinetics in biogeochemical modeling.     

Biological control mechanisms of D-pinitol against powdery mildew in cucumber

D-pinitol is an effective agent for controlling powdery mildew (Podosphaera xanthii) in cucumber. In this study, we determined the mechanisms of D-pinitol in controlling powdery mildew in cucumber plants. We compared P. xanthii development on cucumber leaf surface treated with D-pinitol or water (2 mg ml⁻¹) at different time points after inoculation. The germinating conidia, hyphae, and conidiophores of the pathogen were severely damaged by D-pinitol at any time of application tested. The highest level of suppression of fungal development was obtained at 3 days after inoculation. The contents of chlorophyll, total phenolics, flavonoid, and gallic acid and its derivatives (GAD); the activities of phenylalanine ammonialyase (PAL), polyphenoloxidase (PPO), peroxidase (POX), and superoxide dismutase (SOD); and the expression of the genes encoding for PR-1, peroxidase (POX), lipoxygenase (LOX1), chitinase (Chit1) were higher in the cucumber leaves treated with D-pinitol and inoculated than in the leaves either treated with D-pinitol or inoculated with the pathogen. These results suggest that D-pinitol triggers several plant defense responses in cucumber leading to pathogen suppression and resistance to powdery mildew.