Integrated circuits may be vulnerable to hardware Trojan attacks during its design or fabrication phases. This article is a case study of the design of a Viterbi decoder and the effect of hardware Trojans on a coded communication system employing the Viterbi decoder. Design of a Viterbi decoder and possible hardware Trojan models for the same are proposed. An FPGA-based implementation of the decoder and the associated Trojan circuits have been discussed. The noise-added encoded input data stream is stored in the block RAM of the FPGA and the decoded data stream is monitored on the PC through an universal asynchronous receiver transmitter interface. The implementation results show that there is barely any change in the LUTs used (0.5%) and power dissipation (3%) due to the insertion of the proposed Trojan circuits, thus establishing the surreptitious nature of the Trojan. In spite of the fact that the Trojans cause negligible changes in the circuit parameters, there are significant changes in the bit error rate (BER) due to the presence of Trojans. In the absence of Trojans, BER drops down to zero for signal to noise rations (SNRs) higher than 6 dB, but with the presence of Trojans, BER doesn’t reduce to zero even at a very high SNRs. This is true even with the Trojan being activated only once during the entire duration of the transmission. The entry of connected technologies into the realms of Internet of Things (IoT) and cyber physical systems (CPS) has made it imperative for communications systems to be protected from possible threats. These threats can arise from both software externals and hardware internals. While considerable emphasis is being given to software level threats, in this work we focus on the hardware level threats. The hardware of a communication system can be compromised if its design is exposed so that it can be modified or duplicated. This allows an adversary to deteriorate the performance of a communication system and expose the system to attacks. This makes understanding of the hardware level threats significant. In this work, we focus on the effect of one such threat called hardware Trojans, on coded communication systems that use a Viterbi decoder as the error correcting unit.Design of efficient coder–decoder for error control has received increased interest in recent years. This is due to the fact that all digital transmission and storage requires error control strategy to ensure reliability. Information symbols from a source are encoded by the addition of controlled redundancy. Convolutional codes and block codes are the broad classification of error control codes. An error control decoder makes the best estimate of the transmitted codeword by making use of the redundancy added at the encoder. The transmitted codewords are encoded information symbols that are subject to errors, in the process of transmission through noisy communication channels. These transmitted codewords can be decoded with as low bit error rate (BER) as possible for transmission rates upto the channel capacity . The bloIn the current scenario of integrated circuits (ICs) manufacturing, a globalized business model has emerged where ICs are manufactured in foundries that are distributed in various parts of the world. A hardware Trojan is a malicious stealthy modification that leads to malfunctioning of the system . Such moHardware Trojans can be inserted at different stages of IC design cycle, while the most prevalent phases are design and fabrication. Likewise, Trojans can be realized at different levels of IC design abstraction and can be designed to get triggered internally by specific states of the system, or externally through any communication medium. The former can be stealthy based on the occurrence of the problem states, while the latter will be untraceable in test phase because it is not triggered internally. Regarding the effect of Trojans on the affected system, they are generally designed by the adversary to change functionality or leak sensitive information or deny service during critical instances or compromise the communication system and reduce the reliability of the design .There are numerous post manufacturing techniques for detecting Trojans but a single technique is difficult to be devised for detecting Trojans universally. Side channel and logic testing form the two classical Trojan detection techniques . In thesTrojans are generally modeled for the specific design of interest that they intended to disrupt. Examples of Trojan benchmark circuits aimed at infecting systems like advanced encryption system, serial interface RS232, Ethernet MAC, 8051 and PIC microcontrollers can be found at . These cChannel decoders are a quintessential part of any coded communication system. They are soft targets for Trojan attacks and can be embedded with malicious blocks for the following reasons: .They have a direct interface with the outside world that make them susceptible to being hijacked.They process noisy information that makes it impossible for a even a perfectly functional decoder to be successful all the time. Hence a Trojan affected system may easily claim false failures and masquerade its real purpose.Brute force approach of running all test cases to identify malicious activity is not practical with even a medium size block length since the number of input and output combinations will be huge.In spite of the fact that channel decoders are highly susceptible to Trojan attacks, the effects of Trojans on them hasn’t been explored in literature. Viterbi algorithm is widely used for decoding convolutional codes since it achieves maximum likelihood estimate of the convolutionally coded transmitted sequence . A low BThe article is organized as follows. The Viterbi decoder section details the reader about the Viterbi algorithm with a suitable example. This is followed by the section on the hardware design of the decoder for FPGA implementation. Results from simulation and FPGA implementation of the decoder are discussed after that, followed by the section on the design of the Trojans. Results and discussions on the Trojan based design are presented and the article then concludes with references to possible future work.n encoded output in a  convolutional code depends on the k present input blocks as well the m past input blocks. A memory m sequential circuit is used for realizing the convolutional encoder. The trellis diagram of the rate half, m = 2, convolutional encoder is shown in The The state transitions and the outputs reached in response to changes in input are represented in the state diagram. In the trellis diagram the same information is described in stages which represent the different time instants. With states representing the past memory contents of the encoder and branches representing the state transitions, the trellis path traced by the Viterbi decoder is a sequence of branches. The input message bit corresponding to each branch in the sequence of branches represent the decoded message sequence. By adding terminating zeros to the message sequence it is ensured that the decoder always starts in an all-zero initial state for decoding a message sequence. A detailed example showing the decoding procedure may be found in the The Viterbi decoder was designed in Verilog and implemented on a Xilinx Zybo-Z7010 board. The noise added encoded message is stored in the input BRAM and data is transferred to the decoder block through the input interface. The interface logic unit consists of a counter and an eight bit shit register as shown in It reads the data byte-wise from the BRAM and transmits two bits per clock cycle to the decoder block for further processing. At every clock cycle two LSB bits are shifted out to the branch metric unit (BMU) block. After every four clock cycles, the subsequent BRAM location is read and processed similarly.The BMU calculates the Hamming distance between the received frame and the branch word while the PMU performs add-compare-select (ACS) calculations as described in During every clock cycle, BMU calculates the eight branch metrics corresponding to the two transitions of each of the four states. The branch metrics are then passed on to the PMU that updates each state with the least path metric corresponding to each of the states and also stores the corresponding path leading to it in the form of a “decision bit,” one for each of the four states at every time instant. It can be seen from the trellis diagram in Survivor path memory is designed as a single port BRAM that stores the decision bit values of all states at each of the time instants. The structure of the SPMU is the same as the trellis structure shown in Since the encoded message is terminated with zeros, the final state of the system would be the zero state and hence TBU can be safely initialized to start the decoding from the zero state. After decoding all the 4,096 bits, decode_done signal is activated to trigger the output interface. It is to be noted that it takes around 4,096 clock cycles for the forward tracing, 4,096 clock cycles for traceback and few cycles are required for synchronization.The output interface consists of a first-in-first-out (FIFO) buffer of size 8 × 512  to store the decoded data and the baud generator and transmitter modules of UART to transmit the data serially on to a PC. The UART is designed with a frame length of 10  and works at a baud-rate of 9,600 bps. The data from the decoder is first stored in the FIFO and is transmitted when an external request for data transfer is enabled. The FIFO is designed using a BRAM of size 8 × 512. FIFO is chosen to be one byte wide to enable byte wise data transfer to the UART transmitter easily. The UART transmitter contains the baud generator that generates the UART clock corresponding to a baud rate of 9,600 bps. To generate this clock from the master clock, we use a clock divider whose value can be obtained by using Since the master clock of the FPGA board (Zybo-Z7010 ) is 125 MHz, we need to design a clock divider of value given by Rounding off to the nearest highest integer, we use a clock divider of value 814 to generate the UART clock and data is transmitted out serially to the PC through a RS232 interface.A coded communication system is set up as detailed in the introduction section, using MATLAB. 4 K message bits are generated randomly and encoded using a convolutional encoder of rate 1/2 to generate 8 K encoded bits. Binary phase shift keying modulation is used to modulate the encoded message stream and the resulting data sequence is transmitted through an additive white gaussian noise channel of varying signal to noise rations (SNRs). The received sequences are demodulated and stored as inputs for the Viterbi decoder.The Viterbi decoder was first designed using a behavioral model in MATLAB and then a synthesizable RTL design was done in Verilog. The original encoded message sequence (without noise addition) is given as input to the decoders and the output was verified to be the original message sequence. This establishes that the decoders are functionally correct. Having verified the functionality of the designs using the encoded message as the inputs, the noise-added sequences are given as inputs and the BERs are computed.The RTL design of the decoder was synthesized on to a Zybo board that is built using Z-7010, a member of Xilinx Zynq-7000 family. Z-7010 is designed using Xilinx all programable system-on-chip architecture, that integrates a Xilinx 7-series FPGA along with an ARM based Cortex-A9 processor. In our work, we have not used the ARM processor for generating test inputs but instead, the test vectors are generated from MATLAB as briefed above and a single port BRAM is initialized with these input test vectors. The decoded output bits are first stored in another BRAM and then sent by the UART transmitter to the PMOD pins of the Zybo board. This transmitted data is driven through a UART-to-USB translator (PL2303) and the serial bits are captured on the PC using Real Term serial terminal .Functional verification of the implemented design was done in the same manner as the simulated design. The encoded message bits without noise addition were given as the input and the decoded bits were compared with the original message sequence. It was verified that the decoded and input message bits matched successfully. For calculating the decoder performance, the decoder was fed with noisy data of different SNRs.1Since the BER is plotted on a log scale, it is not possible to indicate the zero values on the plot. Also, there is a perfect overlap of all the plots, thus demonstrating the equivalence of the behavioral, RTL and the implemented models.In this work, we propose the design of three possible Trojans and study how their stealthy presence may affect the system performance.In the PMU, it is expected that the comparator identifies the least path metric path and correspondingly store a “1” or “0” to indicate either of the paths to be traversed during trace back. The proposed Trojan Decision-bit flipping, when enabled, flips the decision bits thus causing the trace back unit to proceed in an incorrect decoding path. The hardware model of the Trojan circuit is shown in The decision bit is inverted when Trojan is enabled and the SPMU gets populated with an incorrect value. This causes the lower metric path to be discarded instead of the higher path metric, thus resulting in possible erroneous decoding.During trace back, the decision bits from the SPMU is read and a path is chosen based on whether the stored value is a “0” or a “1”. The traceback path modification Trojan inverts this logic and changes the state transitions, thus making it proceed in the wrong path. When the Trojan is enabled, the transitions are made to differ from the original state transitions resulting in erroneous decoding.In the output interface, when the decoded data is being written into the shift register, normally it will be right shift operation. But when the Trojan is enabled this operation will be reversed and performs left_shift thus sending erroneous data to the transmitter. This can be achieved by the use of multiplexers that can alter the shift direction based on the Trojan enable signal. The effectiveness of the designed Trojans can be gauged by their stealthy nature and by their propensity to degrade the performance of the infected system.Trojans by nature are stealthy in nature and are difficult to detect. To verify if the proposed Trojan models are stealthy enough to evade detection, the difference in the area and power dissipated due to the Trojan insertion are calculated. It can be seen from the power and area utilization results that in the worst case, there is a difference of only 4 mw of on-chip power (difference of 3.1%), and only four extra LUTs (change of 0.5%) due to the Trojan insertions. This establishes its stealthy nature, thus qualifying them as effective Trojans.To analyze how effectively the Trojans disrupt the natural decoding process, the Trojans are triggered at random time instants and the decoded bits are analyzed. Generally, Trojans are designed to activate surreptitiously in order to go unnoticed. Hence the triggering was done only once during the entire decoding process and the effect of this triggering is observed and the resultant BER is calculated.The effect of the Trojans may be quantified by the increase in the BERs of the infected decoder. It can be seen that in the absence of Trojans, the BER drops down to zero for SNRs greater than 6 dB, but with the Trojans being active, the BER doesn’t reduce to zero even for high SNRs. Thus the Trojans leave a distinctive BER signature (high BER). Among the three Trojans, the BER signature is highest for TBU Trojan and lowest for PMU Trojan, with the shift modification Trojan producing a BER signature in between the other two.It is also interesting to note that the difference in the performances between the Trojan free design and the design with Trojans is negligible in the low SNR regions but the difference is prominent in the high SNR regions. Also at some low SNR conditions, the performance of the Trojan affected system is slightly better than the unaffected system. This scenario is possible since, in the low SNR regions, the data itself is noisy and erroneous. During the bit flipping or the state transition or the shift direction modifying actions of the Trojans there exists a possibility that few erroneous bits are converted to correct bits, thus providing a reverse effect on the system. The possibility of this kind of behavior, along with the fact that Trojans are stealthy make it difficult to conjure effective Trojan detection schemes. To counter such situations, the current focus of researchers is to neutralize them apart from detecting the Trojans .decision bit flipping, traceback path modification and shift direction changing Trojans—and their effect on the decoding efficiency of a Viterbi decoder. The Trojans degrade the performance of the decoder, causing it to have a high BER. But, it is to be noted that high BER can also arise in a system due to high noise in the channel. Hence in situations where the channel’s noise characteristics are unknown, the presence of these Trojan can’t be inferred purely from the BER signature. It needs to be augmented with other Trojan detection schemes to correctly infer the presence of Trojans.The study proposes stealthy Trojans—In this work, we have designed a FPGA based implementation of a Viterbi decoder and presented possible effects of hardware Trojans on coded communication systems. Three unique threat models are developed and tested on the Viterbi decoder which is popular for its low BER performance. However, we show that the presence of the proposed Trojans affect the efficiency of the Viterbi decoder by increasing the BER. The stealthiness of the proposed Trojans is also established. Using the proposed threat models, we envision to test their effects on complex systems like CPS and IoT which rely on efficient communication channels.With the wide application of convolution codes in various SNR scenarios, the results of the implemented system play a significant role in emphasizing the need for efficient Trojan detection schemes. It is envisaged that apart from BER signature analysis, other Trojan detection and neutralizing schemes will be explored for the proposed Trojans.10.7717/peerj-cs.250/supp-1Supplemental Information 1An example for viterbi decoding is illustrated with an trellis diagramClick here for additional data file.10.7717/peerj-cs.250/supp-2Supplemental Information 2This contains data generated for variious SNR. For each SNR, the original message, encoded message, the received data with noise added and the final decoded message. The .coe files for the encoded and received data are also provided. This can be directly used for loading the FPGA memories. encoded.coe files are used for analysis without noise and the received.coe files are used for analysis with noise.Click here for additional data file.


Fluorescence in situ hybridization (FISH) is a powerful method to visualize the spatial positions of specific genomic loci and RNA species. Recent technological advances have leveraged FISH to visualize these features in a highly multiplexed manner. Notable examples include chromatin tracing, RNA multiplexed error-robust FISH (MERFISH), multiplexed imaging of nucleome architectures (MINA), and sequential single-molecule RNA FISH. However, one obstacle to the broad adoption of these methods is the complexity of the multiplexed FISH probe design. In this paper, we introduce an easy-to-use, versatile, and all-in-one application called ProbeDealer to design probes for a variety of multiplexed FISH techniques and their combinations. ProbeDealer offers a one-stop shop for multiplexed FISH design needs of the research community. Chromatin tracing has been combined with single-molecule RNA FISH to study the association between gene expression regulation and chromatin folding5. Recently, our group reported multiplexed imaging of nucleome architectures (MINA)13, a method that combines chromatin tracing, RNA MERFISH and protein labeling. We used MERFISH and cell boundary labeling to distinguish different cell types in a highly heterogeneous mammalian tissue, and used chromatin tracing and co-immunofluorescence to profile three-dimensional genomic architectures in single nuclei across different length scales and in relation to other nuclear components13. These FISH-based methods greatly advanced the characterization of spatial-omics and their physiological relevance. However, one technical obstacle to the broad adaption of these highly multiplexed techniques is their complex probe design procedure. Here, we introduce ProbeDealer, an easy-to-use application that facilitates probe design for chromatin tracing, RNA MERFISH and sequential single-molecule RNA FISH of individual transcript species.Fluorescence in situ hybridization (FISH) can be used to visualize spatial locations of DNA regions and RNA molecules in a sequence-specific manner. Recently, FISH has been extended with multiplicity to profile chromatin folding pattern and the abundance of numerous transcripts in several methods. These include multiplexed sequential DNA FISH14. These requirements were often fulfilled using OligoArray 2.115 in previous works13, which requires familiarity with UNIX. Unfortunately, OligoArray 2.1 is no longer available for download. Thus, users without a previously installed copy of the software will not be able to use our published workflow. Even with OligoArray 2.1, probe design using our previous workflow can be challenging: Output probes must be filtered through additional rounds of BLAST16 to select specific probes, and must be extended with combinations of readout regions and priming sequences, which requires additional programming. The probe filtering and sequence extension algorithms depend on the probe type. Users need to make multiple changes to our previous MATLAB codes to adapt them for their needs. ProbeDealer simplifies this process by integrating probe design, BLAST, and other modifications into one program, and allows versatile probe design for several multiplexed FISH methods. Its graphical user interface improves user experience and eliminates the need for coding expertise.Both chromatin tracing and RNA MERFISH use a two-stage hybridization procedure. In stage one, a library of oligonucleotide probes termed primary probes targeting all genomic loci or RNA species of interest are simultaneously hybridized to the targets. Each primary probe contains a targeting region that hybridizes to the target, and one or more overhanging readout regions. In stage two, dye-labeled secondary probes with different sequences complementing the readout regions on the primary probes are sequentially hybridized to the sample, imaged, and then bleached or removed. To design the primary probes with high hybridization efficiency and specificity, several requirements need to be met, including proper melting temperature (Tm), GC content, minimal secondary structure, minimal cross-hybridization between probes, and lack of long consecutive repeats of the same nucleotideoligoprop function in the MATLAB Bioinformatics Toolbox. To avoid probes containing stable secondary structures, we apply the rnafold function to identify stem-loop structure on oligos. To reject probes that cross-hybridize with other probes, we perform local sequence alignment between each new candidate oligo and the currently accepted oligos using the swalign function. Of note, to facilitate the probe design process, we provide a set of default probe design parameters in an input Excel spreadsheet (oligoparameter.xlsx) which has been tested in our previous experiments13. Users may provide their customized probe design parameters by editing the Excel spreadsheet.The entire workflow of ProbeDealer is illustrated in Fig. 1, RNA will be eliminated and will not bind probes targeting the same sequences in the genome. To accommodate users who may need to retain RNAs, such as for MINA13, ProbeDealer offers an additional feature to design chromatin tracing probes targeting only the antisense strands of gene regions, and therefore even when transcripts are present, the probes will not hybridize to them. Towards this end, users may enable an “only target antisense strand” option in ProbeDealer to BLAST the oligos that passed the whole-genome BLAST further against the unspliced transcriptome in the plus/plus orientation. If an oligo (or its reverse complement) has no hits in the unspliced transcriptome, the oligo (or its reverse complement) is retained. If it is aligned to one or more entries of the unspliced transcriptome in the plus/plus direction, the probe (reverse complement of the oligo) will bind to the unspliced transcript(s), and thus this oligo is rejected.To ensure hybridization specificity, ProbeDealer then BLASTs accepted oligos against specific genomes and transcriptomes, depending on their intended application. For chromatin tracing, oligos are BLASTed against the whole genome in both directions to find oligos that only have one alignment in the genome. Because chromatin tracing is routinely performed with RNase treatmentIn addition, to avoid chromatin tracing probes cross-hybridizing with RNA FISH probes applied to the same sample, ProbeDealer can optionally avoid exon regions of genes. This feature is important especially for MINA, when chromatin tracing and RNA FISH are combined and may target the DNA and RNA from the same genomic region. To achieve this, all chromatin tracing oligos are further BLASTed against spliced transcriptome. Oligos with alignment(s) to the spliced transcriptomes are rejected.To design probes for multiplexed RNA FISH methods, ProbeDealer BLASTs oligos against the spliced transcriptome, and retain probes that only bind to transcript isoforms of the same gene. For both chromatin tracing and RNA FISH probes, users may choose to output all probe sequences, or define how many probes they want for each input target sequence.13 for chromatin tracing probes, and 16 published default secondary probe sequences8 for MERFISH and sequential single-molecule RNA FISH. Users may modify or add secondary probe sequences in the provided Excel spreadsheet of the ProbeDealer package.After filtering oligos according to their physical properties and genomic and/or transcriptomic specificity, ProbeDealer appends sequences of the secondary probes and additional priming regions to the oligos to generate the final template oligos for probe synthesis. ProbeDealer provides 50 published default secondary probe sequences13; (2) primary probe sequences, which can be ordered individually and used directly in primary hybridization. In the first option, three pairs of primer sequences are provided according to previous publications13, with one pair per probe type , so users may combine multiple template oligo libraries from separate ProbeDealer outputs as sub-libraries in one oligo pool order, and selectively synthesize and amplify each sub-library. In the second option, priming regions are not added, and the primary probe sequences are computationally reverse-complemented from the template oligo sequences. This second option is particularly useful for designing small libraries of sequential single-molecule RNA FISH probes for individual targets.We offer two choices of output format: (1) a template oligo library, which may be ordered as oligo pools for probe synthesis and amplification according to a previously established protocol 7 used in MERFISH encoding to reduce bit sharing among highly expressed genes. The ProbeDealer output includes a fasta file and an Excel spreadsheet that contain the full list of template oligo or primary probe sequences. For MERFISH users, ProbeDealer also generates a code book matching MERFISH targets and MHD4 codes in an Excel spreadsheet. A detailed description of ProbeDealer’s internal probe design parameters and procedures is included in the Methods. A user manual including download links of ProbeDealer and examples is included in Supplementary Note We have packaged ProbeDealer as a MATLAB application for Windows and Mac users, and a standalone application for Windows users. The user panel of ProbeDealer is shown in Fig. 13. Specifically, we performed three test runs using ProbeDealer on a workstation with 32 GB RAM using a single core: (1) designing topologically-associating-domain (TAD)-to-chromosome-scale chromatin tracing probes targeting the central 100-kb regions of 50 TADs on mouse chr19, (2) designing promoter-enhancer-scale chromatin tracing probes targeting 19 consecutive 5-kb regions upstream of gene Scd2 on mouse chr19, and (3) designing RNA MERFISH probes targeting 136 mouse transcripts13. The TAD-to-chromosome-scale chromatin tracing design finished in 23.8 h and consumed 5.97 GB RAM. The fine-scale chromatin tracing and RNA MERFISH design took 6.3 min and 18.5 min, respectively, and consumed RAM of 5.47 GB and 1.74 GB, respectively.To estimate the time and memory usage for ProbeDealer to finish different probe design tasks, we recapitulated several probe design schemes in our previous publication1: (1) designing a probe library targeting 30 TADs on human chr20, which takes 19.5 h; (2) designing a probe library targeting 34 TADs on human chr21, which takes 23.1 h; and (3) designing a probe library targeting 40 TADs on human chrX, which takes 21.3 h. The test runs above can also be performed on a personal laptop with 16 GB RAM.We also tested TAD-to-chromosome-scale chromatin tracing design on human genome. Specifically, we recapitulated three chromatin tracing probe designs reported in a previous publication13 using three packages: ProbeDealer, OligoArray 2.115 and OligoMiner17, and compared the number of probes generated by the packages. The probe count per kb of transcript is 29.3 for ProbeDealer, 18.7 for OligoArray 2.1 and 29.1 for OligoMiner. Therefore, ProbeDealer generates comparable number of probes as OligoMiner, which is higher than OligoArray 2.1. Thus, ProbeDealer and OligoMiner are more suitable for designing probes for shorter transcripts, which may not offer enough probes when OligoArray 2.1 is used. A detailed comparison of probe counts among ProbeDealer, OligoArray 2.1 and OligoMiner can be found in Supplementary Table Given the stringent probe specificity consideration in ProbeDealer, we set out to test whether ProbeDealer can yield sufficient number of probes, which is essential for good signal-to-background ratio especially in RNA FISH. We performed test runs of designing RNA MERFISH probes targeting 136 mouse transcripts probed in our previous workWe further compared these probe design tools in terms of their software purposes, computational resource requirements and time costs, probe design criteria, and user-friendliness in Supplementary Note We hope ProbeDealer will simplify probe design for chromatin tracing, RNA MERFISH, sequential single-molecule RNA FISH and MINA, so these methods may be more easily utilized across the scientific community.16. The human genome fasta file (hg38) was downloaded from UCSC genome browser (https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz). The human transcriptome fasta file (Gencode v34) was downloaded from Gencode (ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_34/gencode.v34.transcripts.fa.gz), and the headers of transcript sequences were trimmed in MATLAB so they only contain Ensembl transcript ID without version variant information . To generate the human unspliced transcriptome, the GTF file of hg38 was downloaded from the UCSC genome browser (https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/genes/hg38.ensGene.gtf.gz), and coordinates of transcript start and end were extracted in MATLAB into a BED file. The human unspliced transcriptome fasta file was generated from the BED file and hg38 genome fasta file using command getfasta in BEDTools18.Human and mouse genomes are provided with ProbeDealer. All BLAST databases were generated by makeblastdb command in NCBI BLAST+https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/mm10.fa.gz). The mouse transcriptome fasta file (Gencode vM25) was downloaded from Gencode (ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M25/gencode.vM25.transcripts.fa.gz). The GTF file was downloaded from UCSC genome browser (https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/genes/mm10.ensGene.gtf.gz). Mouse spliced and unspliced transcriptomes were prepared as described for the human transcriptome.The mouse genome fasta file (mm10) was downloaded from UCSC genome browser  in length, with melting temperature (Tm) not lower than 66 °C, and GC content between 30 and 90%. More than five repetitive nucleotides are excluded (e.g. GGGGGG). If probes form secondary structures, the Tm of concatenated stem sequence should not exceed 76 °C. If they cross-hybridize with other probes, the Tm of concatenated hybridized region should not exceed 72 °C. As recommended in OligoArray 2.015, Tm is calculated via the nearest neighbor method19 under the assumption of 1 mol/liter salt concentration and 1e-6 mol/liter probe concentration. Input sequences are scanned with a 30-nt window from the 5′ end. If the current test oligo is accepted, the window moves 30 nt towards 3′ end to evaluate the next adjacent oligo; otherwise, the window moves 1 nt towards 3′ end for a new test oligo. These parameters can be customized by editing oligoparameter.xlsx in the ProbeDealer package.Default probe design uses parameters reported in Ref.For chromatin tracing probes, oligos are first BLASTed against the genome, and only those with unique alignment in the genome are retained. If the “only target antisense of gene” feature is selected, retained oligos are additionally BLASTed against the unspliced transcriptome. Oligos without alignment to the unspliced transcriptome in plus/plus direction are retained. If an oligos is aligned to the unspliced transcriptome in the plus/plus direction but not in the plus/minus direction, its reverse-complement is retained as a qualified oligo. If the “avoid exon regions” feature is selected, qualified oligos are BLASTed once more against the spliced transcriptome. Probes with alignment to the spliced transcriptome are rejected.For RNA FISH probes, including those for RNA MERFISH and sequential single-molecule RNA FISH, oligos are BLASTed against the transcriptome, and oligos matching the transcripts of multiple genes are rejected.13 are provided, respectively. These sequences are appended to the up- and down-stream of the primary targeting region of oligos so that oligos targeting the same input sequence share the same secondary sequence. If the number of input sequences for chromatin tracing or sequential single-molecule RNA FISH exceeds the number of provided secondary sequences, users should provide additional secondary sequences in the secondary sequence Excel spreadsheets  in the ProbeDealer package.For chromatin tracing and sequential single-molecule RNA FISH probes, 50 and 16 default secondary sequences that were previously validated7 coding scheme, which accepts up to 140 input sequences. The MHD4 codes are rearranged according to bulk gene expression values to avoid bit sharing among highly expressed genes. Each of the probes targeting one transcript carries three out of four secondary sequences that are assigned to that transcript, with one secondary sequence upstream of the primary targeting region of the oligo and two secondary sequences downstream.For MERFISH probes, ProbeDealer provides the Modified-Hamming-Distance-4 (MHD4)13, priming regions are appended to both ends of the oligo sequences. By default, chromatin tracing probes, sequential single-molecule RNA FISH probes and MERFISH probes each have one distinct pair of priming sequences. The default priming sequences are stored in Primers.xlsx in the ProbeDealer package and can be customized by the users. The primary probe synthesis and amplification procedure with the template oligo library and the default primers will reverse complement the template oligo sequences to generate the primary probes13. If users require only the primary probe sequences and plan to order them directly, priming regions will not be added and oligo sequences (including the secondary regions) will be computationally reverse-complemented in the output files.If users want to order template oligos as a pooled library for primary probe synthesis and amplificationScd2 gene on mouse chr19, (3) 30 TADs along human chr20, (4) 34 TADs on human chr21, and (5) 40 TADs on human chrX; and one RNA MERFISH test run, which targeted 136 mouse transcripts. The workstation contained an Intel® Core™ i7-8700 K CPU with 6 cores and 12 logical processors, and 32 GB RAM. One processor was used in all test runs.The following test runs in ProbeDealer was performed with a Dell Precision Tower 3630 workstation: five chromatin tracing test runs, which targeted: (1) 50 TADs on mouse chr19, (2) 19 consecutive 5-kb regions upstream of Scd2 gene locus were downloaded from Ref.13, and converted from mm9 to mm10 by the LiftOver tool in UCSC genome browser with default settings. Mouse mm10 genome assembly was used for both test runs. The genomic coordinates of human chr20, chr21 and chrX TADs were downloaded from Ref.1, and the central 100-kb region were used for probe design. For the three test runs targeting human genome, human hg18 genome assembly was used, instead of the default hg38 genome assembly. For all chromatin tracing test runs, we chose the chromatin tracing design scheme without additional features, and all probes were exported.For chromatin tracing test runs, the coordinates of targeted genomic region of mouse chr19 TADs and sequences upstream of 13, and transcript IDs were converted from Gencode vM24 to Gencode vM25, with one failed transcript conversion. We chose the RNA MERFISH design scheme and exported all probes.For MERFISH test runs, 137 mouse transcript IDs and RNA-seq FPKM values were acquired from Ref.The 136 converted mouse transcripts were first divided into 1-kb fragments, and then supplied to OligoArray 2.1 with the following settings: probe length of 30 nucleotides, GC content between 30 and 90%, melting temperature above 66 °C, no 6 or more identical consecutive bases, no stable secondary structure above 76 °C, no cross-hybridization above 72 °C, and no overlaps between probes. OligoArray 2.1 was executed on Yale High Performance Computing Cluster with one node, one core and 32 GB RAM.After probes were generated from OligoArray 2.1, probes were further BLASTed against mouse transcriptome Gencode vM25 with the same criteria as introduced in ProbeDealer MERFISH mode: probes targeting different transcript isoforms of the same gene were retained, while probes targeting multiple transcripts of different genes were rejected.The same 136 mouse transcripts were applied to OligoMiner blockParse.py with the following settings: probe length of 30 nucleotides, GC content between 30 and 90%, melting temperature above 66 °C, formamide concentration 0%, no 6 or more identical consecutive bases. The output probes were then supplied to structureCheck.py with the following settings: salt concentration 330 mM, hybridization temperature 37 °C. The probes that passed structureCheck were then BLASTed against transcriptome with the same criteria in MERFISH test run with OligoArray 2.1.Supplementary Information 1.Supplementary Information 2.


It is the least degree of any extension of the ‘canonical boolean representation’ of the syntactic monoid. Equivalently, it is the least number of states of any subatomic nondeterministic acceptor. It turns out that essentially all previous structural work on nondeterministic state-minimality computes this measure. Our approach rests on an algebraic interpretation of nondeterministic finite automata as deterministic finite automata endowed with semilattice structure. Crucially, the latter form a self-dual category.We introduce a new measure on regular languages: their


Spermatogenesis is the process by which germ cells develop into spermatozoa in the testis. Sperm protamines are small, arginine-rich nuclear proteins which replace somatic histones during spermatogenesis, allowing a hypercondensed DNA state that leads to a smaller nucleus and facilitating sperm head formation. In eutherian mammals, the protamine-DNA complex is achieved through a combination of intra- and intermolecular cysteine cross-linking and possibly histidine-cysteine zinc ion binding. Most metatherian sperm protamines lack cysteine but perform the same function. This lack of dicysteine cross-linking has made the mechanism behind metatherian protamines folding unclear.Protamine sequences from UniProt’s databases were pulled down and sorted into homologous groups. Multiple sequence alignments were then generated and a gap weighted relative entropy score calculated for each position. For the eutherian alignments, the cysteine containing positions were the most highly conserved. For the metatherian alignment, the tyrosine containing positions were the most highly conserved and corresponded to the cysteine positions in the eutherian alignment.High conservation indicates likely functionally/structurally important residues at these positions in the metatherian protamines and the correspondence with cysteine positions within the eutherian alignment implies a similarity in function. One possible explanation is that the metatherian protamine structure relies upon dityrosine cross-linking between these highly conserved tyrosines. Also, the human protamine P1 sequence has a tyrosine substitution in a position expecting eutherian dicysteine cross-linking. Similarly, some members of the metatherian Planigales genus contain cysteine substitutions in positions expecting plausible metatherian dityrosine cross-linking. Rare cysteine-tyrosine cross-linking could explain both observations. The process in which male germ cells develop into sperm cells is called spermatogenesis. During spermatogenesis, DNA undergoes hypercondensation in order to form a smaller nucleus. This is accomplished through the final replacement of a vast majority of somatic DNA histones (>90%) with one of three nuclear proteins; sperm-specific histones, protamine-like proteins, or protamines . In mammGenetically the family of sperm protamines is highly diverse, being observed across the tree of life. For example, a single species of fish can contain multiple genes for protamine and protamine-like proteins, whereas birds tend to have two identical copies of a single protamine gene . While aSome of the simplest protamines are those of fish. Like most sperm protamines, the sequences of fish protamines are populated with a large percentage of arginine residues. However, fish protamines tend to be under 35 amino acids in length and contain increased frequencies of arginine (approximately 70%) in comparison to their mammalian analogs. The secondary structure of the protamines consist of multiple beta turns, with limited CD, NMR, and fluorescence data, indicating the formation of a possible globular structure , 11.Mammals have a relatively conserved set of protamines, with metatherian mammals having only one protamine gene, while eutherian mammals have two to three varieties. These mammalian sequences tend to start with MARYR at the N-terminus, typically followed by a region containing one or more phosphorylation sites, then a DNA binding region comprised of multiple blocks of arginine residues, and ending with a varied C-terminal region , 12.PRM1 gene. Alignment of the sequences of eutherian mammal sperm protamine P1 have shown the sequences to be relatively conserved. In eutherian sperm protamine P1 sequences, the arginine-rich DNA binding regions are broken up by cysteine residues, which are involved in both inter- and intramolecular disulfide cross-linkings [The eutherian protamine P1 is encoded by the linkings , 13, 14.linkings   is involved in the formation of the thiol cross-linking between and within the protamines and with protecting the sperm cells from oxidative stress due to reactive oxygen species , 18. In In contrast to eutherian sperm protamines, little is known about metatherian sperm protamines, except that metatherian sperm protamines tend to lack cysteine residues with the only exception to this tendency involving species of the Planigale genus . To our MSAs were generated for 145 eutherian sperm protamine P1, 16 eutherian sperm protamine P2, 95 metatherian sperm protamine, and 34 fish protamine sequences, all retrieved from the UniProt knowledgebase and aligned using MUSCLE 3.8.31 , 22. TheFrom the fish protamine MSA  . A propoThe comparison of arginine-lysine frequencies of the differing protamine groups showed that although metatherian sperm protamines and fish protamines have similarly high charged residue frequencies across their entire sequences, metatherian mammals have an increased density of charged residues towards the middle of the sequences. This trend is also found in eutherian sperm protamine P1s where there is a central DNA binding region. The charged residue frequencies of the hypothesized DNA binding regions of eutherian P1 and metatherian sperm protamines were the most similar distributions by a significant factor.Additional deviations are also apparent in the MSAs. One example is the tyrosine substitutions (tyr15cys and tyr16cys) of two positions in human sperm protamine P1, which are expected to be involved in intramolecular dicysteine cross-linking in the N-terminal staple fold. It is possible that cysteine-tyrosine cross-linking preserves the cross-linking function in human sperm protamines with these substitutions. A proposed schematic structure for human sperm protamine P1 with conserved cysteine and tyrosine positions is shown in Fig. Likewise, a counter deviation found in the metatherian alignment is with species of the Planigale genus, for which all species but P. maculata contain cysteine substitutions in a number of highly conserved positions where tyrosine residues are expected  of the UniProt KnowledgeBase (SwissProt/TrEMBL)  to creatThe protamine dataset was then broken down into four homologous groups based on existing UniProt gene name and organism classification annotations. The four groups were eutherian sperm protamine P1, eutherian sperm protamine P2, metatherian sperm protamine P1, and fish protamine. The eutherian sperm protamine P1 group was parsed by collecting all sequences which contained ’Eutheria’ in their organism classification and the gene name of either ’PRM1’ or ’Prm1’. The same approach was performed for the eutherian sperm protamine P2 group, but using the gene name of either ’PRM2’ or ’Prm2’. The metatherian sperm protamine P1 group was parsed by collecting all sequences which contained ’Metatheria’ in their organism classification and with the gene name of either ’PRM1’ or ’Prm1’. The fish protamine group was parsed by collecting any sequence which contained ’Actinopterygii’ in their organism classification and that did not contain the word ’like’ in their description. For the eutherian and metatherian groups only a single entry was allowed per organism per group. If multiple sequences existed in a single group, preference was given to the Swiss-Prot entry since these are reviewed entries. Therefore, each group is composed of orthologous genes, with the exception of the fish protamine group where some species have more than one protamine gene in the group.Sperm protamine P2’s contain multiple post-translational cleavage sites, which lead to the removal of 40% of the amino terminus of these proteins . After pAberrant sperm protamine P2 sequences  caused gapping in an initial MSA and were found to lack significant translational expression in prior literature , 33. AlsA fasta file was generated for each homologous protamine group  and then MUSCLE 3.8.31  was usedRelative entropy (Kullback-Leibler divergence) was used to determine residue conservation scores for each position (column of residues) in the alignment. Relative entropy incorporates background frequencies of amino acids to measure the distance between the amino acid frequency in a position of the alignment versus the background frequencies –37.1\doD(P ∥Q) is calculated for each position in the alignment and uses all 20 of the standard amino acids plus Asx (B) for Asp or Asn, Glx (Z) for Glu or Gln, and Xaa (X) for unknown. P(a) is the frequency of the amino acid in the position. Q(a) is the background frequency of an amino acid. For this analysis, the natural abundance of amino acids determined by the UniProt knowledgebase was used . RelativAdditionally, a weighting was used to deal with the presence of gaps in the alignment. The gap weighting is incorporated by multiplying the calculated relative entropy measure by the percent of non-gap residues in the position.To determine which positions are conserved in the alignment, a conservation score threshold equal to a position entirely composed of arginine residues (∼4.1354) was used. If the gap weighted conservation score was greater than the threshold, the position was determined to be conserved. Methionine residues at the beginning of protein sequences are ignored.The arginine-lysine density of each protamine group was calculated by counting the number of arginine and lysine residues in each protamine sequence in the group. Histidine was left out of the analysis as it is mostly deprotonated at physiological pH. Lysine was included above a simple arginine frequency due to known DNA interactions for lysine residues in a variety of DNA binding proteins, but more importantly, an observed reduction in the severity of lower bound outliers for all protamine groups analyzed. This improvement in the lower bound outliers is greater for lysine than for the inclusion of any other amino acid . It is also important to note that there is an evolutionary trend for the replacement of lysine with arginine suggesting arginine is favored over lysine for the condensation of DNA during spermatogenesis . The quaAdditional file 1 The additional figures and tables referenced within this article are available in the figshare repository, https://doi.org/10.6084/m9.figshare.10292573.v1[92573.v1. The sup


Despite these setbacks, harnessing vascular plasticity provides unique opportunities for anti-cancer combination therapies in particular anti-angiogenic immunotherapy which are yet to reach their full potential.Tumour growth critically depends on a supportive microenvironment, including the tumour vasculature. Tumour blood vessels are structurally abnormal and functionally anergic which limits drug access and immune responses in solid cancers. Thus, tumour vasculature has been considered an attractive therapeutic target for decades. However, with time, anti-angiogenic therapy has evolved from destruction to structural and functional rehabilitation as understanding of tumour vascular biology became more refined. Vessel remodelling or normalisation strategies which alleviate hypoxia are now coming of age having been shown to have profound effects on the tumour microenvironment. This includes improved tumour perfusion, release from immune suppression and lower metastasis rates. Nevertheless, clinical translation has been slow due to challenges such as the transient nature of current normalisation strategies, limited  Cancer and stromal accessory cells co-evolve to foster malignant growth and tumour progression. Among stromal cells, tumour blood vessels have been a major focus in oncology. It has been shown in the early 1970s that the rate of tumour neovascularisation – or angiogenesis – controls tumour growth  triggereCoinciding with the idea of killing blood vessels, an alternative concept, namely tumour blood vessel normalisation, first emerged as a strategy to transform the chaotic angiogenic vasculature into a more orderly anatomy which also reduced metastatic dissemination . Since tPfkfb3  re-establishes endothelial adhesion and overall vessel maturation (Phd2 (prolyl-hydroxylase) or its upstream regulator Siah2 (E3 ubiquitin ligase) normalizes vessels by regulating hypoxia-inducible factor (HIF) availability which increases tumour perfusion and pericyte coverage  . For ins+ lymphocyte trafficking in peripheral lymph nodes and at sites of chronic inflammation. HEVs can arise spontaneously in cancer and are associated with a better patient prognosis , a cell type which is morphologically and functionally distinct from endothelial cells. HEVs are cuboidal in shape and decorated with peripheral node addressin (PNAds) which mediate L-selectinrognosis . Importarognosis , 43, 44.rognosis , 46. Overognosis . While cThe advent of checkpoint inhibitors and other immunotherapeutics has changed the oncology landscape profoundly. Impressively, with combined anti-CTLA4  and anti-PD1  treatment, 60% of metastatic melanoma patients now experience a median survival of 2 years rather than 6 months . HoweverBesides inhibition of VEGF signalling, other diverse strategies have been developed to specifically eliminate physical barriers to effector T cell penetration by targeting vascular and ECM features. For instance, CrossMab A2V (Vanucizumab) normalises angiogenic vessels and also stimulates tumour immunity leading to enhanced anti-PD1 effects . Phase IThus, there is a clear correlation between vessel/stromal remodelling and T cell infiltration . HoweverNormalised tumour vessels have been described in many preclinical studies. However, clinical evidence correlating vessel remodelling with better survival outcomes is still sparse. For instance, treatment with VEGF receptor tyrosine kinase inhibitors enhanced survival in those glioblastoma patients who also showed increased tumour perfusion as measured by MRI . NeoadjuFifty years after Judah Folkman demonstrated the critical role of tumour angiogenesis, blood vessels remain an attractive target in the tumour microenvironment. The focus, however, has shifted from vessel destruction to remodelling in response to evidence demonstrating that vessel normalisation and tumour oxygenation are intertwined and crucial for combination therapies. To date, clinical insights into anti-angiogenesis/vessel normalisation therapies are still mainly based on VEGF/VEGFR inhibition. Yet, even after a decade in the clinic, the mode of action, selection of responsive patient populations, treatment timeline and mechanisms of drug resistance remain largely unresolved. However, more recently, the immuno-modulatory effects of anti-VEGF therapy have highlighted the intimate relationship between tumour blood vessels and anti-cancer immunity leading to ongoing clinical trials combining VEGF and checkpoint blockade . Given tThe author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.National Health and Medical Research Councilhttp://dx.doi.org/10.13039/501100000925 of Australia  and the Cancer Council Western Australiahttp://dx.doi.org/10.13039/501100001170 (APP1098579). R Ganss is supported by a Cancer Research Institute Clinic and Laboratory Integration Program (CLIP) Grant, Tour de Cure Senior Research Grant and a Woodside Energy Fellowship.This work was supported by


Early treatment of severe hyperglycemia involves large shifts of body fluids that entail a risk of hemodynamic instability. We studied the feasibility of applying a new electrolyte equation that estimates the degree of volume depletion and the distribution of infused 0.9% saline in this setting.The new equation was applied to plasma and urinary concentrations of sodium and chloride measured before and 30 min after a 30-min infusion of 1 L of 0.9% saline on two consecutive days in 14 patients with severe hyperglycemia (mean age 50 years). The extracellular fluid (ECF) volume was also estimated based on the volume dilution kinetics of chloride.On day 1, the baseline ECF volume amounted to 11.5 L. The saline infusion expanded the ECF space by 160 mL and the intracellular fluid space by 375 mL. On day 2, the ECF volume was 15.5 L, and twice as much of the infused fluid remained in the ECF space. The chloride dilution kinetics yielded baseline ECF volumes of 11.6 and 15.2 L on day 1 and day 2, respectively. No net uptake of glucose to the cells occurred during the two 1-h measurement periods despite insulin administration in the intervening time period.The electrolyte equation was feasible to apply in a group of hyperglycemic patients. The ECF space was 3 L smaller than expected on admission but normal on the second day. Almost half of the infused fluid was distributed intracellularly. High competence is needed to manage fluid, electrolyte, and insulin therapy in patients with poorly controlled diabetes . The sevWe have developed an equation system based on a fluid challenge with 0.9% saline followed by measurements of sodium and chloride that estimate the severity of the dehydration and also the fluid-induced shift of ECF volume into the cells.The aim of the present study was to explore the feasibility of applying the new equation to a group of patients likely to be volume depleted. Severely hyperglycemic patients were considered suitable for this purpose. They receive 0.9% saline and are monitored with measurements of sodium and chloride for clinical reasons.Our hypothesis was that the calculations would enhance knowledge about the severity of the dehydration and the degree of fluid shifts that occur in the treatment of hyperglycemia. The ECF volume was also calculated with volume kinetic analysis of the plasma electrolyte concentrations over time , which iClinicalTrials.gov NCT02172092 before any patient was enrolled. Each patient gave his/her oral and written consent for participation.An infusion experiment was performed on two consecutive days in 14 fully conscious patients who had been admitted for treatment of poorly controlled diabetes to the intensive care unit (ICU) at the Vrinnevi Hospital in Norrköping, Sweden, between 2014 and 2019. The Regional Ethics Committee of Linköping had approved the protocol (Ref. 2014/123-31), and the study was registered at The patients underwent the first infusion experiment soon after their arrival to the ICU and had the repeat infusion on the next day. Each infusion consisted of 1 L of 0.9% saline over 30 min followed by a free interval of 30 min.Arterial blood was withdrawn on 9 occasions between 0 and 60 min. The samples were analyzed for plasma concentrations of sodium (Na), chloride (Cl), and ionized calcium (Ca) on a Radiometer ABL 800 FLEX blood gas machine  with a coefficient of variation of approximately 1%.The patients had a bladder catheter through which the excreted urine was collected, measured, and sampled at 60 min. Urinary electrolytes were measured on the Cobas 8000. The excretion of glucose and electrolytes was taken as the product of the urine volume and the urinary concentration of the solute in question.Monitoring consisted of pulse oximetry and invasive arterial pressures.t). In the present study, we use time frame from 0 to 60 min.The background to the proposed electrolyte calculation comes from a previously used mass balance equation based on changes in Na , 7. By at should equal the left side of the equation if ∆ICF is zero. Any deviation in Nat from its theoretical value means that ∆ICF is separated from zero.All data relating to Na appear in the nominator and the corresponding data for fluid volumes in the denominator. The measured Nao cannot be assumed to be 20% of the body weight in poorly controlled diabetes, we strived to develop an equation that determines both ∆ICF and ECFo. To solve both unknowns, an equation system was set up that considers the changes in both the Na and Cl concentrations after having infused isotonic saline. Chloride occupies the same physiological space as sodium [Because ECFs sodium , 9, and t and Clt after infusion of sodium chloride are given by:By rearrangement, the equation system holds that the finally arrived Nao must be the same in both equations, the following re-arrangement can be made and let the electrolyte changes after the fluid challenge estimate ECFo:As ∆ICF and ECFt) can be solved as:In turn, ∆ICF that has occurred from baseline to time  /ECFo (dependent variable), by the renal fluid clearance, CL . The change of the ECF volume is then described by the following differential equation [In the kinetic model, fluid is infused in ECFFo) Fig. a. Eliminequation , 5:\doco (as given by the electrolyte equation) and Clo to which the infused amount of chloride was added. Losses of chloride were taken as the total excreted amount at 60 min divided by the area under the curve (AUC) for the dilution of plasma Cl but considering only a fraction of AUC for the time segment up to each time t [t and represented by the symbol v, was given by:Correction for losses and additions of Cl was done by taking the total amount of chloride in the ECF volume as the product of ECFh time t . Hence, t − to). ECFo was the key outcome measure of the subsequent volume kinetic calculation. The kinetic calculations were performed by least-squares regression based on a Gauss-Newton routine, using Matlab version 4.2  [where each of the 9 time segments are denoted (o − Cat)/Cat . A non-compartment model analysis (NCA) was chosen because the maximum dilution occurred with a delay of 10–15 min from the end of the infusion. The Phoenix 8.2 software  was used for the analysis, which was designed as “exploratory” because urinary losses for Ca were not measured. However, these losses are small in response to infusion of 0.9% saline [An exploratory analysis of the dilution of plasma Ca was also performed. The dependent variable was then , and P < 0.05 accepted as statistically significant.Group data are presented as the mean and deviation (SD) and the paired The sample size was not determined by a power calculation as the purpose of the study is the feasibility of estimating the body fluid volumes from changes in electrolytes after infusing 0.9% saline.The 14 patients were aged between 18 and 86  years and had body weight of 71  kg. Eight patients had been given 1 L of buffered Ringer’s solution before admission to the ICU, and 3 had received 6–10 units of insulin. The second infusion took place 20.7 (4.2) h after the first one. The patients had then received a total of 7.0 (1.9) L of crystalloid fluid and 26 (16) units of insulin since their arrival to the ICU.P < 0.02) but had little acute effect on the systemic acidosis. Plasma glucose decreased by 20% (P < 0.001). There was profuse glucosuria that averaged to almost 100 mmol during the hour of study in response to the saline. The glucosuria was only 1/4 as large during the second infusion, when many of the blood chemistry parameters had been restored to be within the normal range.Blood and urine chemistry and hemodynamic data are shown in Table The electrolyte equation was applied for the entire period between 0 and 60 min. The calculation employed the mean plasma concentrations of Na and Cl at 0 and 60 min and the mean excreted fluid volume and amounts of Na and Cl at 60 min.On day 1, the ECF volume before the first infusion was initiated amounted to 11.5 L, the expansion of the ECF space from the infusion was 160 mL, and the expansion of the ICF was 375 mL. The remainder of the 1 L of infused fluid had been excreted as urine.On day 2, the ECF volume before the infusion started was 15.5 L, the expansion of the ECF volume was 377 mL, and the expansion of the ICF was 385 mL.The ECF volume was estimated by volume kinetic analysis based on the dilution of the mean plasma chloride concentration for the 14 patients on the 9 points of measurement. A schematic drawing of the kinetic model is shown in Fig. o on day 1 was 11.6 (0.8) L. On day 2, the size of V amounted to 15.2 (1.1) L.This analysis based on Cl showed that the size of ECFo at steady state on day 1 was 11.7 L. On day 2, the size of V amounted to 14.8 L.The exploratory analysis based on Ca showed that the ECFFigure This study comprises patients who had been admitted to ICU for treatment of poorly controlled diabetes, most of which had developed acidosis. Only few had received modest initial treatment with fluid and insulin before being enrolled in the study. We applied a new mass balance approach based on electrolyte shifts to estimate the distribution of short-term infusions of crystalloid fluid on two consecutive days.The results show that the ECF space had a volume of approximately 11.5 L on admission, which is almost 3 L less than expected based on tracer measurements in healthy individuals [Most of the subsequently infused fluid distributed equally between the urine and the ICF space, whereas only 1/6 hydrated the ECF. On day 2, the deficit in the ECF had been corrected, and perhaps even slightly overcompensated. The electrolyte equation further implicated, in light of a smaller diuretic response to the saline, that the infused volume now distributed equally between the ECF and the ICF.Isotonic (0.9%) saline is a fluid composed to remain only the ECF volume, and the pronounced distribution to the ICF on day 1 may then seem odd. Prior to this study, we believed that administration of insulin would promote relocation of fluid to the ICF. However, not even on day 2 had the insulin reversed the catabolic state and promoted uptake of glucose to the ICF volume. This finding might be due to insulin resistance being maintained due to the hyperglycemic environment “glucose toxicity” .The intracellular distribution of infused fluid seemed to be better explained by the glycosuria. When diabetic hyperglycemia develops, gluconeogenesis and glycogenolysis release glucose that translocates ICF water to the ECF by virtue of osmosis. When this glucose is excreted, the osmotic “power” to hold the translocated fluid in the ECV is lost, and it returns to the cells. The lack of glucose uptake also suggests that glycosuria was the key factor, and perhaps the only one, responsible for the decrease in plasma glucose during the first 24 h of treatment.Intravenous fluid should be the initial treatment of poorly controlled diabetes, and the fluid shifts reported in the present study highlight why they entail a risk of inducing hemodynamic instability , 2. The Bergamasco et al.  have devOur current approach overcomes these shortcomings by analyzing the electrolyte changes induced by a fluid challenge. No radioactive tracer is needed and no assumptions about body fluid volumes or “normal” electrolyte concentrations have to be made. Only two plasma samples and one urinary sample are required for the calculations, and only two electrolyte concentrations need to be analyzed. The measurements, as well as the fluid load, are likely to be implemented for clinical purposes anyway, which makes ethical aspects a less crucial issue.The new electrolyte equation was derived from an existing equation used to estimate ICF distribution of hypo-osmotic irrigating fluid in patients with an assumed normal ECF space , 7. The The new equation is mixture of dilution and electrolyte kinetics and provides information about both the degree of pre-infusion volume depletion and the subsequent fluid shift between the ECF and the ICF during the measurement period. The first parameter, but not the second, can also be obtained by applying volume kinetic analysis to a serial analysis of extracellular electrolytes , 5.o were quite similar to those found by the electrolyte equation. However, the kinetic analysis of Cl may not be considered a control method because some data were used in both approaches, although the kinetic analyses used many more of the performed measurements. By contrast, the volume kinetic analysis based on plasma Ca was completely independent of the electrolyte equation, as 0.9% saline solution does not contain calcium. Unfortunately, the Ca dilution showed a delayed time course that necessitated the use of an alternative kinetic model not planned for when the study started.We used chloride for such a complementary calculation, and estimates of ECFLimitations include that only 1 L of saline was infused, and the changes in the plasma concentrations of sodium and chloride were not large enough to safely overcome the associated measurement errors in individual patients. With the present set-up, we recommend that the equation is applied only to group data. The electrolyte changes should be greater to allow reasonably safe application of the new equation on individual patients. Hence, the electrolyte equation was probably more accurate on day 1 than on day 2 because the 0.9% saline induced greater changes in plasma electrolytes at that time. Infusing a larger volume of saline, 2 L rather than 1 L, might overcome the confounding effect of errors in sampling and analysis of electrolyte concentrations.The electrolyte equation does not take non-osmotic sodium, which is found in the bone, skin, and in the glycocalyx, into account. Olde Engeberink et al. showed that a significant amount of sodium was stored in the body when hypertonic saline was infused rapidly to sodium-depleted volunteers . Howevero for Cl than for Na, although these calculations were not corrected for urinary losses of tracer electrolytes [Limitations also include that plasma sodium but not the plasma chloride is known to have a somewhat lower concentration in the interstitial fluid than in the plasma because most plasma proteins have negative charge (the Donnan effect). Experiments with radioactive Na and Cl show a 3% smaller ECFtrolytes . The reltrolytes .In conclusion, a new mass balance equation based on plasma and urinary electrolytes as well as fluid volume kinetics based on chloride dilutions showed an extracellular fluid deficit of approximately 3 L in patients with poorly controlled diabetes admitted to the ICU. The first liter of 0.9% was distributed mainly to the ICF and the urine. Hydration of the ECF improved when the infusion was repeated on the next day. The decrease in plasma glucose during the first hour of fluid treatment was due to osmotic diuresis.


Habitual school backpack carriage causes neuro-musculoskeletal vertebral, shoulder and hand pain; deviated posture compromised cardiopulmonary function and proprioception.Present a novel literature summary of the influence of backpack carriage associated with deviated cervical posture and compromised pulmonary function.An electronic literature appraisal adopting the Preferred Reporting Items for Systematic Reviews, using Google Scholar, Science Direct, EMBASE, AMED, OVID, PubMed and Sabinet search engines, was instituted during 2009–2019. Key search words: schoolbag, backpack, carriage, cervical posture and children. The quality of the studies was assessed using the Downs and Black Appraisal Scale.p < 0.05). Backpack loads carried varied from 5% – 30% of the participant’s body mass that produced a mean CVA decline of 3.5°.583 records were initially identified which was reduced to 14 experimental and observational studies. A total of 1061 participants were included across the 14 studies, with an average age of 11.5 ± 1.3 years, body mass of 37.8 ± 6.6 kilograms (kg), height of 1.41 ± 0.05 meters (m), backpack mass of 5.2 ± 0.9 kg and percentage backpack mass to child’s body mass of 13.75%. The studies mean rating according to the Downs and Black Appraisal Scale was 76.3%. The average craniovertebral angle (CVA) was 53.9° ± 14.6° whilst standing without carrying a backpack was reduced to 50.4° ± 16.4° when loaded (Backpack carriage alters cervical posture, resulting in smaller CVA and compromised pulmonary function. There is no consensus of the precise backpack mass that initiates postural changes. Girls’ posture begin changes when carrying lighter backpacks as compared to boys of the same age strata. The article also reviewed the kinematic association of the altered backpack load-induced cervical posture that changes thoracic alignment, and its influence on pulmonary function. Furthermore, this is the only commentary that presents clinical evidence as per Mill’s Canons of epidemiology.An electronic, narrative literature surveillance adopting the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) benchmarks was followed . Each of the authors completed the three phases, resulting in a list of studies to be synthesised into the commentary. Divergent views amongst the authors whether to include or exclude a study was resolved by holding a joint review and it was put to vote whether the study should be included, based on the application of the inclusion and exclusion criteria (majority vote dictated decision).Participants were records pertaining to the impact of schoolbag backpack carriage on the student’s cervical posture. Participants of the studies had to be within the age strata of 10–14 years and included both genders. The types of studies that were included in this evaluation were empirical articles and randomised control studies. Relevant themes that emerged included altered CHA, CVA and SSA because of schoolbag carriage and rehabilitative exercises were formulated to resolve the altered cervical posture of children carrying heavy schoolbags.Records and articles preceding to the period prior to 2009, relating to the altered cervical posture of adults carrying backpacks, and those of children older than 15 years were not included in the study. Similarly, backpack studies relating to non-cervical posture, electromyography studies (EMG), non-English papers, meta-analyses, systematic reviews and case reports were excluded, as the authors’ primary aim was to synthesise empirical articles pertaining to the aforementioned topic.n = 6 questions), external validity (n = 3 questions), internal validity (n = 3 questions) and power of significance (n = 1 question) of each publication .Research design: studies were classified as randomised control trial, experimental group with concurrent control, and experimental group without control group.Aim: described the aims of the individual studies.Protocols: reported on how sagittal plane posture was analysed, how measurements of CVA, and in some studies CHA and SSA, were carried out.Intervention: the carrying of school backpack.Findings: reported on the change in sagittal plane posture when children carried school backpacks (loaded phase) versus not loaded phase.The authors completed a search in the Sabinet database under the categorisation Medicine and Health. The preliminary search word used was ‘schoolbag’ that yielded 40 records. Then the subsequent word ‘schoolbag carriage’ was entered that yielded nine records. These records were then reviewed for relevance with respect to title and year of publication and it yielded two records. A similar search strategy was completed with regard to the other search engines.Descriptive statistical analyses including mean and percentages were performed. An inferential statistical paired T-test that compared the change in CVA during the unloaded versus loaded phases of the 14 studies was also completed (with the probability factor set at 0.05). The descriptive analyses involved calculating the sum of the participants in the 14 studies, then calculating their mean age, body mass, height and backpack mass. The backpack mass was then expressed as a percentage relative to the average body mass of the participants. The mean CVA of the participants when loaded  and unloaded (not carrying backpacks) was calculated and compared to determine changes in sagittal plane posture. The studies that employed an exercise intervention to combat the effect of heavy backpack post-test CVA results were omitted from the above calculation of the mean CVA and its subsequent comparison.In order to completely comprehend the cascade of sagittal plane kinematics  when carrying school backpacks, a number of terms will need to be defined. The CHA is created by drawing a horizontal line bisecting the tragus of ear and another line drawn from the tragus to the external canthus of the eye  reviewed the effects of backpack loading on cervical posture, four included interventions  , which yielded an average of 13.75% backpack mass relative to the child’s body mass. The average CVA was 53.9° ± 14.6°, whilst standing without carrying a backpack (unloaded phase) was reduced to 50.4° ± 16.4° when loaded ( < 0.05) . BackpacThe following themes evolved from the literature review:The cascade of kinematic events that result in cervical postural deviation, manifested through diminished CVA.The need to identify the specific percent mass of backpack load that initiates changes in CVA.The strength of the clinical evidence supporting the ill effects of backpack loads which produce altered cervical posture amongst children.The similarity of significant changes observed in the cervical curvature when viewed in the sagittal plane was clearly established because of the uniform adoption of fundamental test to measure the CVA change. This helped to improve the validity of the findings. The primary biomechanical objective was to determine through the measurement of CVA whether the carrying school backpacks alter sagittal plane cervical posture. Empirical literature subsequently revealed that altered CVA is accompanied with altered CHA and SSA. However, a common biomechanical explanation of how these altered cervical angles combined to produce a deviated cervical posture is conspicuously missing in the literature. Individual studies have identified changes in the aforementioned sagittal plane angles, but none of them explain the phenomenon holistically. The current discussion will concentrate on the following three themes: the cascade of kinematic events that result in cervical postural deviations, specific percent mass of backpack loads that initiate change in CVA, and the strength of clinical evidence supporting the ill effects of backpack loads, resulting in altered cervical posture amongst children.Children’s sagittal plane posture was altered when they carried backpacks weighing between 5% – 20% of their relative body mass , Khallaf et al. (n = 50 boys) and Goswami et al. (n = 6 boys). A larger sample is needed in order to validate their findings. The empirical evidence indicates that CVA changes occur when a child carries a backpack, but they differ in opinions as to what percent mass of the backpack load produces a significant CVA change. Chansirinukor et al.  within a sufficient time frame to make a plausible conclusion. A total of 11 studies reported a change in children’s cervical posture once backpacks were carried refer to .Strength of association refers to the clinical significance between the disease  and the intervention (carrying backpacks). Eleven studies indicated a strong association between deviated cervical postures when carrying backpacks , where tConsistency of results refers to the consistent observation of the association between the consequence of the intervention (carrying backpacks) and the disease . The 11 empirical studies reported changes in children’s cervical posture when they carried backpacks . The afoBiological plausibility refers to the clinical explanation of the observed outcome of the intervention regarding diseases. The 11 studies also reported altered CVA, CHA, and SSA, indicating altered cervical posture because of carrying backpacks . StudiesDose response refers to the volume of intervention required to produce a specific outcome on the disease. Evidence indicates that a backpack mass greater than 8% for boys and 4% for girls within the 10–14 years age group produces altered cervical posture.This review was not registered with the The International Prospective Register of Systematic Reviews (PROSPERO) website. The review has identified that heavy schoolbag backpack loads alter the sagittal plane cervical posture, reflected by a diminished CVA. However, this altered cervical posture also impacts the SSA and CHA. There were only four studies that measured SSA and CHA, which reflected altered cervical posture. More investigations need to be conducted to document the changes in these associated kinematic angles. Although literature has identified gender variations relating to the extent of percent backpack mass loads that boys and girls can carry, more investigations are needed to guide international paediatric health associations to draft specific independent gender-specific safe backpack mass loads for boys and girls.independent gender-specific safe backpack mass loads for boys and girls.Children carrying backpacks experience a change in their cervical posture, which might alter their normal day-to-day living and wellness. The child’s CVA and SSA diminish, whilst their CHA increases, thus altering the anterior-posterior curvature of the vertebrae, producing a kypholordotic posture and cervical postural syndrome. The empirical evidence indicates that CVA, CHA and SSA changes occur when a child carries a backpack but they differ in opinions as to what relative percent mass of the backpack loads produce significant CVA, CHA and SSA changes. The relatively precise percent backpack load that produces altered CVA, CHA and SSA associated with neuro-musculoskeletal discomfort and/or pain needs to be identified. It is well established that boys are usually stronger than girls within their age-specific strata, which enables them to carry relatively greater backpack loads in relation to their body mass. The altered cervical posture also poses a threat to the child ventilation. Parents, educators and healthcare professionals should consider the aforementioned literature that limits the percent mass load when children carry backpacks. This aforementioned evidence warrants the international paediatric health associations to prescribe


In contrast, after secondary envelopment in fibroblasts or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plasma membrane. We propose that this M6PR pathway is most utilized in VZV infection and least utilized in HSV1 infection, with PRV’s usage being closer to HSV1’s usage. Supportive data from other VZV, PRV, and HSV1 laboratories about evidence for two egress pathways are included.The long-term goal of this research has been to determine why VZV, when grown in cultured cells, invariably is more cell associated and has a lower titer than other alphaherpesviruses, such as herpes simplex virus 1 (HSV1) or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for the transport of single enveloped viral particles from the  trans-Golgi network to late endosomes) was constrained in infected Pompe cells. When cells were analyzed with two different anti-M6PR antibodies, extensive colocalization of the major VZV glycoprotein gE (known to contain M6P residues) and the M6P receptor (M6PR) was documented in the viral highways at the surfaces of non-Pompe cells after maximum-intensity projection of confocal z-stacks, but neither gE nor the M6PR was seen in abundance at the surfaces of infected Pompe cells. Taken together, our results suggested that (i) Pompe cells lack a VZV trafficking pathway within M6PR-positive large endosomes and (ii) most infectious VZV particles in conventional cell substrates are transported via large M6PR-positive vacuoles without degradative xenophagy to the plasma membrane.The literature on the egress of different herpesviruses after secondary envelopment is contradictory. In this report, we investigated varicella-zoster virus (VZV) egress in a cell line from a child with Pompe disease, a glycogen storage disease caused by a defect in the enzyme required for glycogen digestion. In Pompe cells, both the late autophagy pathway and the mannose-6-phosphate receptor (M6PR) pathway are interrupted. We have postulated that intact autophagic flux is required for higher recoveries of VZV infectivity. To test that hypothesis, we infected Pompe cells and then assessed the VZV infectious cycle. We discovered that the infectious cycle in Pompe cells was remarkably different from that of either fibroblasts or melanoma cells. No large late endosomes filled with VZV particles were observed in Pompe cells; only individual viral particles in small vacuoles were seen. The distribution of the M6PR pathway  or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for the transport of single enveloped viral particles from the trans-Golgi network within small vacuoles to the plasma membrane. In contrast, after secondary envelopment in fibroblasts or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plasma membrane. We propose that this M6PR pathway is most utilized in VZV infection and least utilized in HSV1 infection, with PRV’s usage being closer to HSV1’s usage. Supportive data from other VZV, PRV, and HSV1 laboratories about evidence for two egress pathways are included. A goal of this research has been to determine whether the autophagic flux pathway, which includes the late endosome, is relevant to varicella-zoster virus (VZV) egress after secondary envelopment. There are many conflicting reports in the herpes virology literature about the proviral and antiviral roles of autophagy in the infectious cycle –4. Likew–9–trans-Golgi network (TGN) to the late endosome via a mannose-6-phosphate receptor (M6PR) pathway, in which there is a prominent cation-independent M6PR (CI-M6PR) and a minor cation-dependent M6PR (CD-M6PR). Recently, investigators have discovered a deficiency in autophagic flux in Pompe disease  in Pompe cells. We also wanted to repeat our original data about increased LC3-II after VZV infection of MRC-5 cells as a positive control, because another laboratory had not found a similar response . When peBecause of our extensive autophagy studies during VZV infection using confocal microscopy , we alsoNext, we wanted to compare levels of viral protein expression in Pompe cells and MRC-5 cells by confocal microscopy as a confirmation of the decreased VZV titer in Pompe cells. For this experiment, we selected the VZV structural glycoprotein gE, which is the major type 1 VZV glycoprotein expressed in the early-late phase of the VZV replication cycle . By confWe also examined the monolayers for a VZV capsid protein (open reading frame 41 [ORF 41]) and the VZV gC protein, a true late protein . ExpressBecause we planned to observe infected Pompe cells by transmission electron microscopy (TEM) in order to investigate VZV trafficking pathways, we first needed to observe uninfected Pompe cells by TEM, in part because we were unable to find sufficient examples in the literature. As a control, we reexamined uninfected MRC-5 cells, and as a second control for autophagy inhibition, we reexamined MRC-5 cells treated with BAF. We divided the micrographs into images of four areas of the cell: nucleus, cytoplasm, Golgi apparatus, and cytoplasm with plasma membrane. Compared with uninfected and untreated MRC-5 cells 1 to -4, Next, we examined the nuclei of infected Pompe cells in comparison with the nuclei of infected MRC-5 cells and infected BAF-treated MRC-5 cells. We reasoned that an impairment in VZV nuclear egress in Pompe cells may signal an unanticipated alteration in VZV capsid formation and tegumentation and thereby compromise our ability to directly compare means of egress after secondary envelopment in the cytoplasm. Capsid formation under typical conditions in infected MRC-5 or human melanoma cells is known to lead to aberrant capsids, many of which lack a DNA core , 30. As trans-Golgi network  versus infected MRC-5 cells , we realized that we had found a cell line with a restricted egress pathway never seen before in any other cell line examined by our laboratory since we first looked in 1978 . Our hypAs another control for the specificity of our gradient sedimentation, we also purified adenovirus in the same density gradient. Adenovirus is another double-stranded DNA virus, but adenovirus lacks an envelope (80 nm in diameter). As can be seen, a single band was detected after gradient sedimentation at approximately the same location as that of the VZV capsid band in the Pompe sedimentation , blot e.We have previously proposed that VZV exits the cell by two different pathways, because the results about virus egress published by several laboratories (including our laboratory) over the past decades could not be explained by egress in a single known cellular pathway . When we37–We have observed in prior studies that a greater number of viral particles are produced in human melanoma cells than in MRC-5 cells and that the viral particles egress onto the cell surface in a distinctive pattern called viral highways, although particles are not released in the medium . TherefoFig. 7DTo expand the spatial organization of the organelles within the images, we also analyzed 11 sets of z-stacks (each z-stack contains about 40 images) with Imaris software, which converts each z-stack into a 3D animation, allowing the viewer to see the depth within a cell where the virus-organelle interaction of interest occurs. To this end, each confocal z-stack was analyzed with Imaris 3/4D Image Visualization and Analysis software; the Imaris Coloc module was used to identify colocalization of virus and the M6PR. The areas of colocalization were displayed as both maximum-intensity projections and isosurface renderings . The lenWe carried out the above-described confocal colocalization experiments in VZV-infected melanoma cells because of the distinctive viral highways on the cell surface. As a further positive control, we purified VZV from infected MRC-5 cells by density gradient sedimentation. The infectious virus band was collected from fraction 12 of the gradient and subjected to immunoblotting with rabbit anti-M6PR antibody. The VZV-infected cell lysate prior to sedimentation was added to the immunoblot experiment. As can be seen in After investigating the expression of the M6PR protein, we also examined transcription representing the receptor in uninfected and VZV-infected cells. To this end, we carried out real-time (RT)-PCR measurements of RNAs for VZV ORF 68 (VZV gE) together with loci encoding both forms of the M6PR in MRC-5 cells and Pompe cells. VZV infection of MRC-5 cells was robust by 72 to 96 hpi, as indicated by high levels of expression of VZV gE RNA, although both CD-M6PR and CI-M6PR RNA levels were not significantly changed . VZV inftrans-Golgi network, where the final sugar residues are added  to its defined linear epitope (DK = 1.5 × 10−7 M)  . No cros 10−7 M) .Late endosomes comprise large vacuoles  because they can fuse with one another or with other organelles to form hybrid compartments, sometimes called kiss-and-run fusions . We had VZV exocytosis in the small vacuole pathway in Pompe cells is the alternative pathway that does not involve the M6PR. When we purified viral particles from Pompe cells by density gradient sedimentation, we were able to detect both the VZV gE protein and the Rab6 protein in the virus band. Features of this secretory pathway have been described by the Elliott laboratory and the Enquist laboratory, using HSV1 and PRV, respectively . Both laFinally, we present in The interaction between the M6PR and gE on the viral particle may have been easier to recognize in our experiments because they were observed within the distinctive viral highways on the cell surface by high-resolution confocal microscopy . We haveThere may also be an evolutionary link between the M6PR and HSV1 and VZV glycoproteins. Viral genes within the unique short genome (including both gD and gE) are considered to be late additions to each alphaherpesvirus genome that facilitate and fine-tune adaption to its specific host. There is speculation that the predominant VZV gE protein , which is larger than its HSV1 gE counterpart , has subsumed functions of the small HSV1 gD . Even though VZV and PRV are more closely related to each other than to HSV1, VZV has lost gD, while PRV has retained gD , 68. TogDQ479961) (5 infectious units/ml) or cell-free MRC-5 virus inoculum (104 PFU/ml)  . AdenoviQ479961) , 71. VirQ479961) . Pompe d PFU/ml) . The ino PFU/ml) . Altoget PFU/ml) . To measMonoclonal antibodies against VZV proteins were produced and characterized at the University of Iowa, Iowa City, IA, USA . Rabbit z axis for each x,y position. The methods for scanning electron microscopy of VZV-infected monolayers have been described in detail in prior publications  yielded approximately 3 μg of RNA in 80 μl. Polyadenylated RNA was converted to cDNA using anchored oligo(dT) primers and the SuperScript III first-strand synthesis system for RT-PCR (Invitrogen), to yield approximately 20 ng of cDNA. The samples were subsequently digested with ribonuclease H to remove RNA bound to the cDNA. One microliter of cDNA was added to 25 μl of Applied Biosystems master mix-water, together with approximately 200 nM specific primers (see the primer table in TC) values of duplicate samples were averaged and normalized to an average of 16.0 for GAPDH  to form ΔTC values. Subsequent ΔΔTC values were calculated by differences between averages of VZV-infected ΔTC values with the average uninfected ΔTC values. Data points represent means and standard errors of the means from three independent experiments.Total RNA was extracted from uninfected and VZV-infected fibroblasts or Pompe cells in six-well plates at the given time points using the RNeasy minikit (Qiagen). RNA quality and quantity were assayed by UV spectroscopy using a NanoDrop spectrometer (ThermoFisher Scientific).


The John A. Hartford Foundation and the Association for Gerontology Education in Social Work (AGESW) have worked to develop gerontological social work faculty to address the needs of older adults. This presentation will discuss the role of AGESW’s Pre-Dissertation Fellows Program in the development of social work doctoral students. All participants from the PDFP’s 2010-2016 cohorts received a 38-question online survey via email exploring the program’s impacts on their academic career in teaching, research, mentoring, and support. Forty-five respondents, representing all six cohorts, completed the survey. More than half said the PDFP contributed to their ability to publish research , grow their professional network , and teach . Doctoral programs provided different experiences: mentoring, methodological training, professional development, and peer support. Results suggest the PDFP supplements students’ doctoral programs by connecting students to each other and to national leaders.