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Eusociality is a rare but successful life-history strategy that is defined by the reproductive division of labour. In eusocial species, most females forgo their own reproduction to support that of a dominant female or queen. In many eusocial insects, worker reproduction is inhibited via dominance hierarchies or by pheromones produced by the queen and her brood. Here, we consider whether these cues may act as generic 'environmental signals', similar to temperature or nutrition stress, which induce a state of reproductive dormancy in some solitary insects. We review the recent findings regarding the mechanisms of reproductive dormancy in insects and highlight key gaps in our understanding of how environmental cues inhibit reproduction., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

The following study used 3-T functional magnetic resonance imaging (fMRI) to investigate the neural signature of Kamin blocking. Kamin blocking is an associative learning phenomenon seen where prior association of a stimulus (A) with an outcome blocks subsequent learning to an added stimulus (B) when both stimuli are later presented together (AB) with the same outcome. While there are a number of theoretical explanations of Kamin blocking, it is widely considered to exemplify the use of prediction error in learning, where learning occurs in proportion to the difference between expectation and outcome. In Kamin blocking as stimulus A fully predicts the outcome no prediction error is generated by the addition of stimulus B to form the compound stimulus AB, hence learning about it is "blocked". Kamin blocking is disrupted in people with schizophrenia, their relatives and healthy individuals with high psychometrically-defined schizotypy. This disruption supports suggestions that abnormal prediction error is a core deficit that can help to explain the symptoms of schizophrenia. The present study tested 9 healthy volunteers on an f-MRI adaptation of Oades' "mouse in the house task", the only task measuring Kamin blocking that shows disruption in schizophrenia patients that has been independently replicated. Participant's Kamin blocking scores were found to inversely correlate with Kamin-blocking-related activation within the prefrontal cortex, specifically the medial frontal gyrus. The medial frontal gyrus has been associated with the psychological construct of uncertainty, which we suggest is consistent with disrupted Kamin blocking and demonstrated in people with schizophrenia. These data suggest that the medial frontal gyrus merits further investigation as a potential locus of reduced Kamin blocking and abnormal prediction error in schizophrenia.

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.

Background: Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods., Methods: We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h., Results: Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics., Conclusions: A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine or outpatient implementation. Here, we evaluate a novel microfluidic real-time PCR platform (Advanced Liquid Logic, Research Triangle Park, NC) that is rapid, portable, and fully automated. We enrolled patients with CAP and extracted DNA from nasopharyngeal wash (NPW) specimens using a biotinylated capture probe and streptavidin-coupled magnetic beads. Each extract was tested for M. pneumoniae-specific DNA by real-time PCR on both conventional and microfluidic platforms using Taqman probe and primers. Three of 59 (5.0%) NPWs were positive, and agreement between the methods was 98%. The microfluidic platform was equally sensitive but 3 times faster and offers an inexpensive and convenient diagnostic test for microbial DNA., ((c) 2010 Elsevier Inc. All rights reserved.)

This paper details the development of a digital microfluidic platform for multiplexed real-time polymerase chain reactions (PCR). Liquid samples in discrete droplet format are programmably manipulated upon an electrode array by the use of electrowetting. Rapid PCR thermocycling is performed in a closed-loop flow-through format where for each cycle the reaction droplets are cyclically transported between different temperature zones within an oil-filled cartridge. The cartridge is fabricated using low-cost printed-circuit-board technology and is intended to be a single-use disposable device. The PCR system exhibited remarkable amplification efficiency of 94.7%. To test its potential application in infectious diseases, this novel PCR system reliably detected diagnostic DNA levels of methicillin-resistant Staphylococcus aureus (MRSA), Mycoplasma pneumoniae , and Candida albicans . Amplification of genomic DNA samples was consistently repeatable across multiple PCR loops both within and between cartridges. In addition, simultaneous real-time PCR amplification of both multiple different samples and multiple different targets on a single cartridge was demonstrated. A novel method of PCR speed optimization using variable cycle times has also been proposed and proven feasible. The versatile system includes magnetic bead handling capability, which was applied to the analysis of simulated clinical samples that were prepared from whole blood using a magnetic bead capture protocol. Other salient features of this versatile digital microfluidic PCR system are also discussed, including the configurability and scalability of microfluidic operations, instrument portability, and substrate-level integration with other pre- and post-PCR processes.

Using ovule clearing, more than 33,600 ovules of Rhododendron nuttallii T. W. Booth (Ericaceae) were examined for megagametophyte and early postfertilization stages at daily intervals from anthesis until 3 weeks after pollination. Pretreatments with amyloglucosidase to digest integumentary and gametophyte starch and Stockwell's bleach to remove tannins from the integumentary epidermis were necessary. Ovules were cleared by a combination or modifications of Herr's four-and-a-half or Stelly's hemalum-methyl salicylate techniques and were observed using differential interference contrast optics. The method proved suitable for large-scale quantitative studies of ovule development and fertilization. A general protocol is suggested as a starting point for ovule clearing studies.

In Rhododendron spp. and Ledum groenlandicum a callose wall is laid down around the zygote in the first 2 days after fertilization. The periodic acid/Schiff-positive, aniline blue-fluorescence-positive callosic wall is initiated adjacent to the degenerating synergid, extends to cover the entire zygote surface, and remains visible during the initiation of embryogeny as the zygote elongates before the first proembryonal division. Unfertilized ovules show eventual callose deposition in the ovule wall cells during senescence in undeveloped abscising pistils, but show no development of callose within the embryo sac. Possible roles of a zygote special callose wall are discussed.