Microsystems for biomechanical measurements

The use of microtechnology to make biomechanical measurements allows for the study of cellular and subcellular scale mechanical forces. Forces generated by cells are in the few nanoNewton to several microNewton range and can change spatially over subcellular size scales. Transducing forces at such small size and force scales is a challenging task. Methods of microfabrication developed in the integrated circuit industry have allowed researchers to build platforms with cellular and subcellular scale parts with which individual cells can interact. These parts act as transducers of stresses and forces generated by the cell during migration or in the maintenance of physical equilibrium. Due to the size and sensitivity of such devices, quantitative studies of single cell and even single molecule biomechanics have become possible. In this review we focus on two classes of cellular force transducers: silicon-based devices and soft-polymer platforms. We concentrate on the biomechanical discoveries made with these devices and less so on the engineering behind their development because this is covered in great detail elsewhere.     

Diffusion-weighted and perfusion MR imaging for brain tumor characterization and assessment of treatment response

Diffusion-weighted magnetic resonance (MR) imaging and perfusion MR imaging are advanced techniques that provide information not available from conventional MR imaging. In particular, these techniques have a number of applications with regard to characterization of tumors and assessment of tumor response to therapy. In this review, the authors describe the fundamental principles of diffusion-weighted and perfusion MR imaging and provide an overview of the ways in which these techniques are being used to characterize tumors by helping distinguish tumor types, assess tumor grade, and attempt to determine tumor margins. In addition, the role of these techniques for evaluating response to tumor therapy is outlined.     

High‐resolution magnetic resonance angiography in the mouse using a nanoparticle blood‐pool contrast agent

High‐resolution magnetic resonance angiography is already a useful tool for studying mouse models of human disease. Magnetic resonance angiography in the mouse is typically performed using time‐of‐flight contrast. In this work, a new long‐circulating blood‐pool contrast agent—a liposomal nanoparticle with surface‐conjugated gadolinium (SC‐Gd liposomes)—was evaluated for use in mouse neurovascular magnetic resonance angiography. A total of 12 mice were imaged. Scan parameters were optimized for both time‐of‐flight and SC‐Gd contrast. Compared to time‐of‐flight contrast, SC‐Gd liposomes (0.08 mmol/kg) enabled improved small‐vessel contrast‐to‐noise ratio, larger field of view, shorter scan time, and imaging of venous structures. For a limited field of view, time‐of‐flight and SC‐Gd were not significantly different; however, SC‐Gd provided better contrast‐to‐noise ratio when the field of view encompassed the whole brain (P < 0.001) or the whole neurovascular axis (P < 0.001). SC‐Gd allowed acquisition of high‐resolution magnetic resonance angiography (52 × 52 × 100 micrometer³ or 0.27 nL), with 123% higher (P < 0.001) contrast‐to‐noise ratio in comparable scan time (～45 min). Alternatively, SC‐Gd liposomes could be used to acquire high‐resolution magnetic resonance angiography (0.27 nL) with 32% higher contrast‐to‐noise ratio (P < 0.001) in 75% shorter scan time (12 min). Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

Reevaluation of the role of LIP-1 as an ERK/MPK-1 dual specificity phosphatase in the C. elegans germline

The fidelity of a signaling pathway depends on its tight regulation in space and time. Extracellular signal-regulated kinase (ERK) controls wide-ranging cellular processes to promote organismal development and tissue homeostasis. ERK activation depends on a reversible dual phosphorylation on the TEY motif in its active site by ERK kinase (MEK) and dephosphorylation by DUSPs (dual specificity phosphatases). LIP-1, a DUSP6/7 homolog, was proposed to function as an ERK (MPK-1) DUSP in the Caenorhabditis elegans germline primarily because of its phenotype, which morphologically mimics that of a RAS/let-60 gain-of-function mutant (i.e., small oocyte phenotype). Our investigations, however, reveal that loss of lip-1 does not lead to an increase in MPK-1 activity in vivo. Instead, we show that loss of lip-1 leads to 1) a decrease in MPK-1 phosphorylation, 2) lower MPK-1 substrate phosphorylation, 3) phenocopy of mpk-1 reduction-of-function (rather than gain-of-function) allele, and 4) a failure to rescue mpk-1–dependent germline or fertility defects. Moreover, using diverse genetic mutants, we show that the small oocyte phenotype does not correlate with increased ectopic MPK-1 activity and that ectopic increase in MPK-1 phosphorylation does not necessarily result in a small oocyte phenotype. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the C. elegans germline. Our results caution against overinterpretation of the mechanistic underpinnings of orthologous phenotypes, since they may be a result of independent mechanisms, and provide a framework for characterizing the distinct molecular targets through which LIP-1 may mediate its several germline functions.

Extracellular signal-regulated kinases (ERKs) are a group of serine/threonine protein kinases and classical members of mitogen activated protein kinases (MAPKs). The ERK MAPKs are terminal enzymes of a highly conserved three-tiered kinase signaling cascade, the RAS–ERK pathway (1, 2). Extracellular stimuli, including growth factors and insulin signaling induce the sequential activation of RAS–ERK pathway that orchestrates a wide range of cellular processes such as gene expression, proliferation, differentiation, and apoptosis to regulate tissue and organismal homeostasis (Fig. 1A) (1–3). Because the ERK MAPK signaling pathway regulates a myriad of developmental processes for controlled and ordered execution of the pathway, ERK activity is tightly monitored in space and time (4). MEK (also known as MAPK/ERK kinase) phosphorylates ERK at threonine and tyrosine residues (TEY motif), thus activating its function (1). Active ERK is then inactivated by dual specificity MAPK phosphatases (MKPs or DUSPs) that remove the phosphate residues. Together, MEK and DUSPs shape the magnitude, duration, and spatiotemporal profile of ERK activity (1, 4–6).Open in a separate windowFig. 1.lip-1 mutants are defective in pachytene exit and oocyte formation. (A) Schematic view of the conserved LET-60 (RAS)–MPK-1 (ERK) pathway showing that the regulation of ERK/MPK-1 activation depends on upstream kinase cascade and dephosphorylation depends on DUSPs. (B) Schematic view of a hermaphroditic C. elegans germline displaying the spatiotemporal nature of MPK-1 activation. The germline is oriented in a distal (*) to proximal direction from left to right. Proliferative PZ cells are in the distal region, capped by the distal tip cell (DTC). Germ cells enter meiotic prophase at the transition zone (TZ), followed by progression through different stages of meiotic prophase. The “loop region” is the anatomic bend in the U-shaped gonad. The −1 marks the oldest oocyte at the proximal end. Active MPK-1 is visualized by a specific dpMPK-1 antibody in two distinct regions of the germline: midpachytene, termed as zone 1, and proximal few oocytes, termed as zone 2. The intensity of the color (red) correlates with strength of MPK-1 di-phosphorylation. (C) Predicted activation of MPK-1 in the absence of DUSP: either distal to zone 1, called “precocious” activation, or in the late-pachytene/early-diplotene region (anatomically in the loop region), called “ectopic” activation. (D–I) Differential interference contrast microscopy images of germlines from indicated genotypes, age, and temperature to visualize germline morphology. The loop region is on the right in the photographs and oocytes on the ventral side. Oocytes are numbered from proximal to distal polarity (toward loop). The most proximal oocyte is labeled as −1. Arrowheads indicate oocytes, and arrows indicate pachytene-stage germ cells. (J) Quantification of germlines of the indicated genotypes, with pachytene-progression–defective phenotypes expressed as a percentage. (K–P) Dissected DAPI-stained germlines of the indicated genotypes (mid-L4 + 24 h at 25 °C) displaying germline morphology. Insets are magnified views of germ cell(s) at the proximal gonad (after loop region). The dissected germlines are oriented with the distal on the left (*) to proximal on the right of the photograph, according to the meiotic progression. Arrowheads indicate oocytes, and arrows indicate pachytene germ cells. The total number of germlines (n) analyzed per genotype is indicated in each panel (scale bars, 25 μm).The Caenorhabditis elegans oogenic germline, like most complex biological systems, displays a controlled spatiotemporal pattern of ERK (MPK-1 in C. elegans) activity (7–11). Active MPK-1, as assayed using an antibody that detects dual phosphorylated MPK-1 at threonine and tyrosine of the TEY motif (7, 12), is visualized in midpachytene (termed as zone 1 of activation). However, MPK-1 is dephosphorylated, and thus, its activity is very low in the late-pachytene and early-diplotene region of the germline, which corresponds to the anatomic “loop” of the C. elegans U-shaped gonad (Fig. 1B). MPK-1 phosphorylation is again visible in the proximal diakinesis oocytes (termed as zone 2) in a hermaphroditic germline (7–9). Zone 2 activation is mediated by a secreted sperm signal (major sperm protein, or MSP), which antagonizes the VAB-1 Ephrin receptor (13). Thus, zone 2 activation is absent in C. elegans females, which do not produce sperm (7). In a wild-type oogenic hermaphroditic germline, active MPK-1 has not been visualized in the distal germline, from the progenitor zone (PZ) to midpachytene, and is very low in the loop region of the germline. Because total MPK-1 protein is expressed throughout the germline (8), the striking spatiotemporal activation pattern of MPK-1 observed using the dual-phosphospecific antibody suggests localized activation and inactivation of MPK-1 through MEK and DUSPs.In the oogenic hermaphroditic germline, the phenotypic consequences of MPK-1 activation are complex. In genetic mutants of the mpk-1 pathway, changes to the MPK-1 activation pattern along the spatiotemporal axis, as well as alterations to signal strength, produce distinct phenotypes. For example, a complete loss of MPK-1 activity in a null allele causes the oogenic germ cells to arrest in early- to midpachytene (8, 14). In the absence of MPK-1 activity, the germ cells fail to launch the apoptotic program because they do not progress into midpachytene, the stage in which meiotic checkpoint activation culls errors (9, 15). Reduction of MPK-1 signal strength using temperature-sensitive (ts) alleles, however, produced different phenotypes depending on the time at which MPK-1 activation was reduced during oogenic development. These mpk-1(ts) germlines exhibit increased apoptosis (due to higher levels of meiotic asynapsis defects; Ref. 11), a pachytene-progression defect in which pachytene-stage cells linger and are observed in the loop region, and fewer oocytes with an increased size relative to wild-type animals (8). Conversely, in RAS/let-60 gain-of-function mutants, the spatiotemporal pattern of MPK-1 activation is different from the wild-type in two regions: 1) in midpachytene, the germline displays “precocious” activation of MPK-1, and 2) the loop region exhibits “ectopic” MPK-1 activation (Fig. 1C). These animals, unlike the wild-type, display multiple small oocytes (8). Because of the striking increase in oocyte number in the RAS/let-60 gain-of-function mutants, an increase in oocyte number has been considered as a readout for MPK-1 activation. Mutants displaying multiple small oocytes are thus interpreted to be a consequence of increased MPK-1 activity.The C. elegans genome has 29 predicted DUSPs, of which LIP-1 (lateral signal induced phosphatase-1) bears homology with mammalian DUSP6/7 (16, 17). Genetic evidence suggested that loss of lip-1 negatively regulates MPK-1 during somatic vulval development (17). In vitro, in mammalian Cos-1 cultured cells, Myc-tagged LIP-1 protein was shown to dephosphorylate mammalian ERK1/2 (16). Coupled with the homology to mammalian DUSPs, the authors concluded that LIP-1 functions as an MPK-1 DUSP in vivo. In the C. elegans germline, immunofluorescence staining using an anti-LIP-1 antibody showed that the total protein is expressed from the proximal one-third of the PZ region and throughout the pachytene as membrane-associated bright puncta (18). LIP-1 was proposed to function as an MPK-1 DUSP, in the germline, from two lines of evidence (18), which we reevaluated based on the reasoning outlined below. First, in the prior report, the authors showed that in a feminized germline, which does not produce any sperm signal, loss of lip-1 led to an increase in phosphorylated MPK-1 in zone 2 (Fig. 1B). However, in the absence of the sperm signal, MPK-1 cannot be phosphorylated in zone 2 to begin with (7, 13) (Fig. 1A). In this context, inactivation or absence of a DUSP (LIP-1, in this case) should not lead to an increase in the level of phosphorylated MPK-1 since it was never phosphorylated. Second, the authors observed that loss of lip-1 led to ectopic (loop region) MPK-1 activation in hermaphrodites coupled with an increase in oocyte numbers. The authors interpreted this phenotype to be similar to that of RAS/let-60 gain-of-function mutant germlines (18). However, recent work has revealed that the increased oocyte production in RAS/let-60 gain-of-function animals is due to the “precocious” activation of MPK-1 in the early-pachytene, rather than the “ectopic” MPK-1 activation in the loop region (Fig. 1C) (11). Together, these two lines of reasoning led us to reinvestigate the role of LIP-1 as an MPK-1 DUSP in the C. elegans germline and to determine where in the germline spatiotemporal axis LIP-1 might function to regulate oocyte formation, using cytology, genetics, and phenotypic analyses.Contrary to what was previously published (18), our results show that 1) precocious or ectopic MPK-1 activity is not detected in the absence of lip-1—in fact, we found that loss of lip-1 led to lower MPK-1 activation; 2) loss of lip-1 fails to rescue the pachytene-progression and fertility defects observed upon reducing mpk-1 function; and 3) germlines with loss of lip-1 displayed an mpk-1 loss-of-function–like oocyte phenotype, rather than a gain-of-function–like oocyte phenotype, and 4) led to lower MPK-1 substrate phosphorylation. Moreover, we show that mutants in other genes, such as ooc-5 (human ortholog of torsinA AAA+ ATPase), also exhibit multiple small oocytes (19, 20) but do not present with ectopic MPK-1 activity, suggesting that increased oocyte number is not invariably equivalent to, or due to, increased MPK-1 phosphorylation. In support of this, we observed that loss of rskn-1 (human ortholog of RPS6KA, ribosomal protein S6 kinase A), which results in increased ectopic activation of MPK-1 in the loop region of the germline, does not exhibit increased oocyte numbers. This finding demonstrates that ectopic MPK-1 activation does not necessarily cause oocyte numbers to increase. Finally, in wild-type C. elegans diplotene oocytes, the synaptonemal complex (SC) central proteins are removed from the long arm of the chromosome axis to allow for accurate chromosome segregation (21). However, RAS/let-60(ga89ts) gain-of-function mutants have been shown to retain the SC central proteins on the long arm (10). Nadarajan et al. (10) reported that loss of lip-1 also leads to retention of the SC central protein to the long chromosomal arm and proposed that this was because of an increase in MPK-1 activation. We found that while the SC central element proteins are retained on the long arm of the chromosome in diplotene oocytes in both RAS/let-60(ga89ts) gain-of-function and lip-1 mutant oocytes, they are not retained in the rskn-1 mutant germlines, which display increased MPK-1 activation in oocytes. Thus, the retention of the SC central proteins in lip-1 mutant germlines likely occurs through MPK-1–independent mechanisms, suggesting that multiple regulatory processes, both independent of and dependent on ectopic MPK-1 phosphorylation, control SC disassembly. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the context of the C. elegans germline and may have multiple other targets through which it mediates its several germline functions.     

Robust designation of meiotic crossover sites by CDK-2 through phosphorylation of the MutSγ complex

Crossover formation is essential for proper segregation of homologous chromosomes during meiosis. Here, we show that Caenorhabditis elegans cyclin-dependent kinase 2 (CDK-2) partners with cyclin-like protein COSA-1 to promote crossover formation by promoting conversion of meiotic double-strand breaks into crossover–specific recombination intermediates. Further, we identify MutSγ component MSH-5 as a CDK-2 phosphorylation target. MSH-5 has a disordered C-terminal tail that contains 13 potential CDK phosphosites and is required to concentrate crossover–promoting proteins at recombination sites. Phosphorylation of the MSH-5 tail appears dispensable in a wild-type background, but when MutSγ activity is partially compromised, crossover formation and retention of COSA-1 at recombination sites are exquisitely sensitive to phosphosite loss. Our data support a model in which robustness of crossover designation reflects a positive feedback mechanism involving CDK-2–mediated phosphorylation and scaffold-like properties of the MSH5 C-terminal tail, features that combine to promote full recruitment and activity of crossover–promoting complexes.

Sexually reproducing organisms rely on proper chromosome segregation during meiosis to produce gametes with a complete genome. During meiotic prophase I, chromosomes pair and undergo crossover recombination with their homologous partners. This process, together with sister chromatid cohesion, leads to the formation of physical linkages between the homologs and enables their separation during meiosis I. Defects in crossover formation are disastrous, leading to miscarriages and congenital disorders, such as Down syndrome (1).Meiotic recombination initiates with the generation of programmed DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme Spo11 (2). DSBs are resected to yield two 3′-end single-stranded DNA (ssDNA) overhangs, which are rapidly coated by RecA recombinases Dmc1 and Rad51. This nucleoprotein filament then seeks out homology and invades a homologous template, forming a metastable single-end invasion intermediate (D-loop) (3). The invading strand primes DNA synthesis and extends the D-loop. If the extended D-loop is captured by ssDNA on the other side of DSBs in a process known as second-end capture, a double Holliday junction (dHJ) forms (4). While dHJs can be resolved biochemically as either crossovers or non–crossovers (5), during meiosis, the majority of dHJs are specifically resolved as crossovers through the activity of MutLγ (MLH1-MLH3) (6–8) or other structure-selective endonucleases. Although a multitude of DSBs are generated during meiotic prophase, strikingly few are ultimately selected to become crossovers. Early recombination intermediates pare down in pachytene until each homolog pair receives at least one crossover, while the majority of DSBs are repaired as non–crossovers via synthesis-dependent strand annealing (9). However, how meiotic DSBs are chosen to become crossovers remains poorly understood.Throughout eukaryotes, crossover recombination is primarily controlled by a group of proteins collectively termed “ZMM” (10). Notably, homologs of the yeast RING (Really interesting new gene) domain protein Zip3 [ZHP-1, ZHP-2, ZHP-3, and ZHP-4 in Caenorhabditis elegans (11–14), Drosophila Vilya and Narya/Nenya (15, 16), Hei10 in Arabidopsis (17), and Hei10 and RNF212 in mammals (18, 19)] initially localize as abundant foci or long stretches along the synaptonemal complex (SC) but eventually concentrate at crossover sites in late pachytene (20). These SUMO or ubiquitin ligases appear to promote crossover designation by stabilizing the ZMM proteins at crossover sites while removing them from other recombination intermediates (13, 14, 18, 19, 21, 22). Although many meiotic proteins are shown to be SUMO modified (23), key targets of the Zip3 family proteins remain largely unknown.The meiosis-specific MutS homologs MSH4 and MSH5 form a heterodimeric MutSγ complex and play essential roles in crossover formation in diverse eukaryotes (24–31). MutSγ localizes to recombination intermediates as numerous foci but ultimately accumulates at sites that are destined to become crossovers (32, 33). Biochemical analyses using recombinant MSH4 and MSH5 have shown that MutSγ recognizes single-end invasion intermediates and HJs in vitro (34, 35). HJs activate the ATP hydrolysis of MutSγ and promote the exchange of bound ADP for ATP, inducing the formation of a sliding clamp that dissociates from HJs (35, 36). By iterative loading and embracing DNA duplexes within a dHJ, MutSγ is thought to stabilize crossover–specific recombination intermediates (33, 35). In addition, MutSγ recruits and activates the resolvase activity of MutLγ, enabling biased processing of dHJs into crossovers during meiosis (37, 38).A genetic screen in C. elegans identified a cyclin-like protein COSA-1 as a component essential for processing meiotic DSBs into crossovers (39). The mammalian ortholog CNTD1 was subsequently identified (40), and both COSA-1 and CNTD1 have been shown to localize to crossover sites (39, 41, 42). In the absence of COSA-1/CNTD1, MutSγ components persist as numerous foci in pachytene, and crossover formation is eliminated or severely compromised (33, 40), demonstrating a crucial role of COSA-1/CNTD1 in converting early recombination intermediates into crossovers. Because both COSA-1 and CNTD1 are members of the cyclin family, it is plausible that they form a complex with a cyclin-dependent kinase (CDK) and regulate the recombination process through phosphorylation.Several lines of evidence have suggested that CDK2 might be a relevant kinase partner for CNTD1. CDK2 interacts with CNTD1 in yeast-two hybrid assays (41, 42) and localizes to interstitial chromosome sites (43, 44) in a CNTD1-dependent manner (40). Reduced CDK2 activity leads to a failure in crossover formation, while a hyperactive form of CDK2 causes an increased number of MLH1 foci (45). However, due to its requirement at telomeres in tethering chromosomes to the nuclear envelope, deletion of CDK2 leads to severe defects in SC assembly between paired homologs (synapsis) and pachytene arrest (46–49). Further, while a full-length CNTD1-specific protein of the excepted size was detected (using CNTD1 antibodies) in one study (42), a short CNTD1 isoform that cannot interact with CDK2 was the predominant isoform detected in another study (using hemagglutinin antibodies in Cntd1^FH/FH mice with an epitope tag sequence inserted into the endogenous Cntd1 locus) (41), raising questions regarding the extent to which CDK2 and CNTD1 might act as functional partners. Thus, it has been difficult to determine the role of CDK2 in crossover recombination. Moreover, key meiotic targets of CDK2 have not yet been identified.We reasoned that CDK-2, the C. elegans homolog of CDK2, might also localize and function at crossover sites. However, global knockdown of C. elegans CDK-2 by RNA interference leads to cell cycle arrest of mitotically proliferating germ cells (50), thereby precluding the analysis of its requirement during meiotic prophase. To overcome this limitation and establish the meiotic function of CDK-2, we use the auxin-inducible degradation system to deplete CDK-2 from the adult germline, demonstrating that CDK-2 partners with COSA-1 to promote crossover formation during C. elegans meiosis. Moreover, we identify MSH-5 as a key substrate for CDK-2 and provide evidence that CDK-2 and COSA-1 partner to promote crossover designation through phosphorylation and activation of the MutSγ complex.     

Secondary developmental glaucoma in eyes with congenital aphakia

Purpose:To describe the clinical spectrum and management of glaucoma in congenital aphakia.Methods:The demographics and clinical spectrum of eyes with congenital aphakia with and without glaucoma were compared, and management outcomes of congenital aphakia cases with glaucoma were studied retrospectively between April 2000 and June 2020.Results:There were a total of 168 eyes (84 subjects) with a diagnosis of congenital aphakia, of which 29 eyes of 18 subjects were diagnosed with glaucoma. Corneal opacity was the presenting complaint in 26/29 eyes with glaucoma and 139/139 eyes without glaucoma. The (interquartile range (IQR)) horizontal corneal diameter was 10.5mm (IQR, 9.0-12.5) and 8mm (IQR, 5-10) in eyes with and without glaucoma (P = 0.01), respectively. The median (IQR) axial length was 17.5mm (IQR, 13.5-19.5) and 15mm (IQR, 14-16) mm in eyes with and without glaucoma (P = 0.03), respectively. Nineteen eyes with glaucoma had adequate intraocular pressure (IOP) control with one medication. Three eyes underwent transscleral diode cyclophotocoagulation and maintained IOP without medications. Three eyes underwent trabeculectomy and trabeculotomy, trabeculectomy followed by penetrating keratoplasty, and trabeculectomy, respectively, of which two eyes became phthisical. At the last follow-up, the median (IQR) IOP was 14 mm Hg (IQR, 14-17) Hg. The median (IQR) follow-up duration was 4.53 months (IQR, 2.03- 48.06).Conclusion:One-fifth of the eyes with congenital aphakia had secondary developmental glaucoma. The corneal diameter and axial lengths were higher in the eyes with glaucoma compared to eyes without glaucoma. Medical management is the preferred short-term mode of IOP control. Transscleral cyclophotocoagulation may be preferred over surgical intervention.